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PubMed2001

Topology of the human skeletal muscle chloride channel hClC-1 probed with hydrophilic epitope insertion.

To investigate the membrane topology of the skeletal muscle chloride channel ClC-1, we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAPEDPED) epitope tags into nine predicted extra- and intracellular loops along the channel protein. Functional integrity of the modified proteins was tested by measuring the chloride currents conducted by these channels expressed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7, D8D9 or D11D12 did not alter channel function significantly, whereas insertion into D3D4, D5D6, D9D10 and D10D11 led to loss of function. Intra- or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected with the functional epitope-inserted constructs. Intact cells stained for the epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these linkers face the extracellular side of the membrane. No conclusions could be drawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5), a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.

Cell Line, Transformed↗
PubMed2000

A novel reverse-genetic approach (SIMF) identifies Mutator insertions in new Myb genes.

We have developed a new strategy designated SIMF (Systematic Insertional Mutagenesis of Families), to identify DNA insertions in many members of a gene family simultaneously. This method requires only a short amino acid sequence conserved in all members of the family to make a degenerate oligonucleotide, and a sequence from the end of the DNA insertion. The SIMF strategy was successfully applied to the large maize R2R3 Myb family of regulatory genes, and Mutator insertions in several novel Myb genes were identified. Application of this technique to identify insertions in other large gene families could significantly decrease the effort involved in screening at the same time for insertions in all members of groups of genes that share a limited sequence identity.

Amino Acid Sequence↗
PubMed1997

The identification of a (CGG)6AGG insertion within the CGG repeat of the FMR1 gene in Asians.

We have evaluated the structure of the CGG repeat within the FMR1 gene of an Asian population and found the most common size of the repeat to be 29 and 30 with a minor population of 36 repeats. We have isolated and sequenced DNA containing the 36 repeats and found the basis sequence to be (CGG)9AGG(CGG)9AGG-(CGG)6AGG(CGG)9; with a (CGG)6)AGG insertion, designated as 9A9A6A9. Of 144 Asian chromosomes, 11 (8%) had sequences with this insertion. Six different variations of the basic sequence were observed in the population: 9A9A6A2A9, 9A9A6A11, 9A9A16, 9A9A15, 8A9A6A6A9, and 11A6A6A9. All but one of the chromosomes with the insertion had the haplotype of DXS548/ FRAXAC1: 194/D suggesting that the sequences with the 6A insertion arose from a single ancestral allele. We have not observed the insertion in the FMR1 gene of Caucasians or Native Americans. The (CGG)6AGG insertion may be unique to Asians.

Alleles↗
PubMed2001

The Basques according to polymorphic Alu insertions.

Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups.

Alu Elements↗
PubMed2001

Germ line insertion of mtDNA at the breakpoint junction of a reciprocal constitutional translocation.

Constitutional chromosomal translocations are relatively common causes of human morbidity, yet the DNA double-strand break (DSB) repair mechanisms that generate them are incompletely understood. We cloned, sequenced and analyzed the breakpoint junctions of a familial constitutional reciprocal translocation t(9;11)(p24;q23). Within the 10-kb region flanking the breakpoints, chromosome 11 had 25% repeat elements, whereas chromosome 9 had 98% repeats, 95% of which were L1-type LINE elements. The breakpoints occurred within an L1-type repeat element at 9p24 and at the 3'-end of an Alu sequence at 11q23. At the breakpoint junction of derivative chromosome 9, we discovered an unusually large 41-bp insertion, which showed 100% identity to 12S mitochondrial DNA (mtDNA) between nucleotides 896 and 936 of the mtDNA sequence. Analysis of the human genome failed to show the preexistence of the inserted sequence at normal chromosomes 9 and 11 breakpoint junctions or elsewhere in the genome, strongly suggesting that the insertion was derived from human mtDNA and captured into the junction during the DSB repair process. To our knowledge, these findings represent the first observation of spontaneous germ line insertion of modern human mtDNA sequences and suggest that DSB repair may play a role in inter-organellar gene transfer in vivo. Our findings also provide evidence for a previously unrecognized insertional mechanism in human, by which non-mobile extra-chromosomal fragments can be inserted into the genome at DSB repair junctions.

Base Sequence↗
PubMed2005

Insertion loads of the X STOP interspinous process distraction system designed to treat neurogenic intermittent claudication.

An interspinous process implant has been developed to treat patients suffering from neurogenic intermittent claudication secondary to lumbar spinal stenosis. As most patients who suffer from spinal stenosis are over the age of 50 and may have weaker bones, it is imperative to know how bone mineral density (BMD) correlates with lateral spinous process strength. The study was undertaken to characterize the lateral failure loads of the spinous process, correlate the failure loads to BMD, and compare the failure loads to the loads required to insert an interspinous process implant. Spinous process lateral failure loads were assessed, correlated to BMD, and compared to the loads required to insert an interspinous process implant. Mean spinous process failure loads were significantly greater than the lateral insertion load of the interspinous process implant. There was a significant relationship between the BMD and spinous process failure load. The technique used to insert the interspinous implant poses little risk to spinous process failure. There is ample margin of safety between the insertion loads and spinous process failure loads. The significant relationship between BMD and spinous process failure load suggests that patients with lower BMD must be approached with more caution during the implant insertion procedure.

Biomechanical Phenomena↗
PubMed2000

Accuracy of pedicle screw insertion with and without computer assistance: a randomised controlled clinical study in 100 consecutive patients.

We performed a randomised controlled study to assess the accuracy of computer-assisted pedicle screw insertion versus conventional screw placement under clinical conditions. One hundred patients scheduled for posterior thoracolumbar or lumbosacral pedicle screw instrumentation were randomised into two groups, either for conventional pedicle screw placement or computer-assisted screw application using an optoelectronic navigation system. From the computer-assisted group, nine patients were excluded: one because of an inadequate preoperative computed tomography study, seven because of problems with the specific instruments or the computer system, and one because of an intraoperative anesthesiological complication. Thus, there were 50 patients in the conventional group and 41 in the computer-assisted group, and the number of screws inserted was 277 and 219, respectively. There was no statistical difference between the groups concerning age, gender, diagnosis, type of operation performed, mean operating time, blood loss, or number of screws inserted. The time taken for screw insertion was significantly longer in the computer-assisted group. Postoperatively, screw positions were assessed by an independent radiologist using a sophisticated CT imaging protocol. The pedicle perforation rate was 13.4% in the conventional group and 4.6% in the computer-assisted group (P = 0.006). Pedicle perforations of more than 4 mm were found in 1.4% (4/277) of the screw insertions in the conventional group, and none in the computer-assisted group. Complications not related to pedicle screws were two L5 nerve root lesions, one end plate fracture, one major intraoperative bleeding and one postoperative death in the conventional group, and one deep infection in the computer-assisted group. In conclusion, pedicular screws were inserted more accurately with image-guided computer navigation than with conventional methods.

Adult↗
PubMed2007

Implications of deep electrode insertion on cochlear implant fitting.

Using long Med-El Combi40+ electrode arrays, it is now possible to cover the whole range of the cochlea, up to about two turns. Such insertion depths have received little attention. To evaluate the contribution of deeply inserted electrodes, five Med-El cochlear implant users were tested on vowel and consonant identification tests with fittings with first one, two, and up to five apical electrodes being deactivated. In addition, subjects performed pitch-ranking experiments, using loudness-balanced stimuli, to identify electrodes creating pitch confusions. Radiographs were taken to measure each electrode insertion depth. All subjects used each modified fitting for two periods of about 3 weeks. During the experiment, the same stimulation rate and frequency range were maintained across all the fittings used for each individual subject. After each trial period the subject had to perform three consonant and three vowel identification tests. All subjects showed deep electrode insertions ranging from 605 degrees to 720 degrees. The two subjects with the deepest electrode insertions showed significantly increased vowel- and consonant-identification performances with fittings with the two or three most apical electrodes deactivated compared to their standard fitting with all available electrodes activated. The other three subjects did not show significant improvements in performance when one or two of their most apical electrodes were deactivated. Four out of five subjects preferred to continue use of a fitting with one or more apical electrodes deactivated. The two subjects with the deepest insertions also showed pitch confusions between their most apical electrodes. Two possible reasons for these results are discussed. One is to reduce neural interactions related to electrodes producing pitch confusions. Another is to improve the alignment of the frequency components of sounds coded by the electrical signals delivered to each electrode to the overall pitch of the auditory perception produced by the electrical stimulation of auditory nerve fibers.

Adult↗
PubMed2006

Use of an ultrathin gastroscope to allow endoscopic insertion of enteral wallstents without fluoroscopic monitoring.

Self-expanding metallic stents are useful in relieving tumoral obstruction in the gastrointestinal tract. Endoscopic insertion is usually made through the working channel of a therapeutic endoscope. Fluoroscopy during insertion is thought to be mandatory in most cases. Endoscopists sometimes encounter problems in using or accessing fluoroscopy facilities. This study describes a method to insert, under certain circumstances, enteral Wallstents using only endoscopic control. An ultrathin gastroscope is used to pass severe tumoral strictures and place a guide wire beyond the stenosis. The ultrathin gastroscope is removed leaving the guide wire in place, which is then inserted in a retrograde fashion into a therapeutic colonoscope, allowing insertion of through-the-scope stents. Successful insertion was achieved in 5 malignant gastric outlet obstructions and in 6 rectosigmoid tumoral obstructions. In conclusion, in some cases using an ultrathin endoscope to place a guide wire beyond the stricture can be useful for endoscopic placement of Wallstents without fluoroscopy.

Adenocarcinoma↗
PubMed2002

Effect of skin disinfection with octenidine dihydrochloride on insertion site colonization of intravascular catheters.

BACKGROUND: We investigated the efficacy of two commercially available, alcohol-based antiseptic solutions in decontaminating the insertion site of central lines. One solution contained the bispyridine octenidine dihydrochloride. PATIENTS AND METHODS: Inpatients receiving either a central venous catheter (CVC) or a peripherally inserted central catheter (PICC) were alternately assigned to different skin disinfection regimens at the insertion site: (A) 0.1% octendine dihydrochloride with 30% 1-propanol and 45% 2-propanol, (B) 74% ethanol with 10% 2-propanol. Quantitative skin cultures were obtained from the insertion site at predetermined intervals. RESULTS: A total of 60 patients received 12 CVCs and 47 PICCs (no significant difference with respect to gender, age and catheter type). In total, 90 cultures were assessed in each group. The median colony-forming unit (cfu) counts per 24 cm(2) (group A vs B) were 2,270 vs 2,950 before, 20 vs 40 following and 860 vs 1,210 24 h after catheter insertion, respectively. A statistically significant difference in the efficacy of skin decontamination was seen between groups in culture set (3) and in the difference between culture sets (2) and (3) (Wilcoxon rank sum test). CONCLUSION: Octenidine/propanol appears to be more effective than alcohol (ethanol/propanol) alone in reducing microflora of the skin at the PICC/CVC insertion site over a 24-h period.

1-Propanol↗
PubMed1983

The nature of structural defects associated with velamentous and marginal insertion of the umbilical cord.

Previous studies have documented an increased incidence of structural defects in association with velamentous and marginal insertion of the umbilical cord. Evaluation of 4,677 consecutive placentas at the University of California (San Diego) Medical Center revealed 454 marginal insertions and 72 velamentous insertions. An increased incidence of structural defects was found only in association with a velamentous cord insertion. Delineation of the structural defects according to their developmental pathogenesis revealed that the majority were due to deformation of a normally formed part. These data and the finding of a disproportionate number of twins with a velamentous insertion suggest that competition for space at the implantation site leads to both the velamentous insertion of the cord and the associated structural defects.

Abnormalities, Multiple↗
PubMed1983

Use of T4 DNA polymerase replacement synthesis for specific labeling of plasmid-cloned inserts.

A strategy employing T4 DNA polymerase replacement synthesis is described whereby only the insert portion of recombinant plasmids are radioisotopically labeled. Prior purification of the inserted DNA is not required. The recombinant plasmid is first digested with one or more restriction endonucleases selected to cleave the vector segment into fragments at least 30% shorter than the insert DNA segment. This mixture of fragments is then digested by the T4 DNA polymerase-associated 3' exonuclease in the absence of deoxynucleoside triphosphates (dNTPs) for a length of time which allows complete degradation of all fragments shorter than the insert. The remaining insert DNA, which is now partially single-stranded, is then resynthesized by addition of dNTPs, one or more of which is labeled. The resulting DNA is full length, double-stranded, and unnicked. The strategy is widely applicable, and reliably and reproducibly yields DNA of high specific activity. We have used this method to label more than 15 cloned inserts ranging in size from 3.2 to 25 kilobases.

Cloning, Molecular↗
PubMed1987

Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior.

The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.

Amino Acid Sequence↗
PubMed1985

Expulsions in immediate postpartum insertions of Lippes Loop D and Copper T IUDs and their counterpart Delta devices--an epidemiological analysis.

In this paper, an epidemiological analysis was performed, using an international data set, exclusively on the expulsion problem associated with postpartum IUD insertions. The inserter's experience in postplacental insertions is probably an important determining factor for IUD expulsions. Immediate insertions (within 10 minutes after placental delivery) are possibly associated with lower expulsion rates than later insertions (eg. two to 72 hours after placental delivery) during the woman's postpartum hospitalization. No significant differences were detected between the standard Lippes Loop D and Copper T IUDs and their counterpart Delta devices specifically designed for postpartum use, between the two types of Delta device or between the hand and inserter methods. A case-control analysis also did not detect any significant association between IUD expulsions and mild complications occurring or management performed during the third stage of labor and delivery. The practical implications of these findings, the methodologic problems of this analysis and future research strategies are also discussed.

Adult↗
PubMed1994

Effect of prophylactic antibiotics on morbidity associated with IUD insertion: results of a pilot randomized controlled trial. IUD Study Group.

The efficacy of administering an antibiotic prior to IUD insertion to reduce the risk of introducing an upper genital tract infection during the procedure has not yet been established. Two double-blind randomized studies conducted in Africa comparing a 200 mg prophylactic dose of doxycycline with a placebo did not conclusively identify a reduced risk of post-insertion pelvic inflammatory disease (PID). A clinical trial of comparable design is currently under way in the US. This multi-site trial will evaluate whether use of an antibiotic prior to insertion reduces the risk of IUD removal for all medical reasons, including upper genital tract infection, within the first three months after insertion. This paper reports on the pilot phase of this study, which was designed to test the protocols and data collection instruments in advance of the full-scale clinical trial. A total of 447 prospective IUD (TCu-380A) users were randomly assigned to receive either a 200 mg dose of doxycycline or a placebo one hour before IUD insertion. 3.6% (8/219) of participants who received the antibiotic had the device removed for medical reasons (infection, bleeding, cramping, etc.) within three months post-insertion compared to 4.5% (10/223) of participants who received the placebo. This reduction in the removal rate was not statistically significant given the limited size of the pilot study (RR = 0.81; 95% CI 0.28-2.29). Only two subjects, one from each treatment group, met the diagnostic criteria for acute PID. The overall three-month retention rate was 91.8% for the antibiotic group and 89.7% for the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

A second isoform of chicken brush border myosin I contains a 29-residue inserted sequence that binds calmodulin.

Chicken brush border myosin I (CBB-MI) is a single-headed, nonfilamentous, myosin-like mechanoenzyme which, as isolated, has 3 mol of calmodulin (CAM) 'light chains' bound per mole of 119 kDa heavy chain. We have isolated a partial cDNA clone for CBB-MI that encodes the C-terminal approximately 35 kDa of the heavy chain. The sequence of this clone is identical to that of an authentic, near-full-length CBB-MI cDNA clone reported recently, except for an 87-bp/29-residue insertion occurring approximately 32 kDa from the C-terminus. This insert, which is probably generated by an alternate splicing event, is expressed in brush border as part of a message of the size predicted for the CBB-MI heavy chain, although the steady state level of this transcript is approximately 8-fold lower than for transcripts lacking the insert. 125I-CAM overlays of this cDNA clone (expressed as a trpE fusion protein in E. coli) indicate that it binds one more calmodulin than does a second cDNA clone that lacks the 29-residue insert. A synthetic peptide corresponding to the insert sequence binds tightly to CAM-Sepharose, demonstrates a shift and enhancement of fluorescence in the presence of CAM, and binds CAM in solution with a KD of 190 nM (in 100 mM KCl). We conclude that a second, low-abundance isoform of CBB-MI contains an additional (and possibly fourth) CAM binding site as a result of a 29-residue peptide that is inserted into the tail domain by an apparent alternate splicing event.

Amino Acid Sequence↗
PubMed1995

Stress governs tissue phenotype at the femoral insertion of the rabbit MCL.

The cells in the midsubstance portion of skeletal ligaments typically have elongated shapes, but where ligaments insert into bone the cells appear very rounded and the tissue phenotype is that of fibrocartilage. Between the midsubstance and the insertions there is a gradient in cell shape and tissue phenotype that has been hypothesized to reflect a gradient of mechanical stresses. To test this hypothesis, cell shapes (an index of tissue phenotype) were quantified in the central part of the femoral insertion of the rabbit medial collateral ligament by computer-assisted histomorphometry. Morphometric measurements were correlated with the mechanical stresses and strains in the central part of the insertion as predicted by finite element analysis. Throughout the ligament the direction of the predicted principal tensile stresses coincides with the direction of the collagen fibers which curve from the midsubstance to meet the femur at nearly right angles. Principal compressive stresses also occur within the ligament: the highest are localized near the bone; the lowest in the midsubstance. The areas with the roundest cells correspond to the areas with the highest principal compressive stresses in the model; the areas with the flattest cells correspond to the areas with the lowest compressive stresses in the model. A correlation between cell shape and mechanical stresses suggests that physiological loading of the MCL is important for the maintenance of tissue phenotype throughout this insertion. We theorize that the cells in ligament insertions adapt to the prevailing local mechanical environment.

Adaptation, Physiological↗
PubMed1995

The effect of variable relative insertion orientation of human knee bone-ligament-bone complexes on the tensile stiffness.

In order to evaluate the contribution of the knee ligaments to restrain joint motions, knowledge about their structural properties is required. Due to the variable relative insertion orientation of the ligaments during knee motion, however, different fiber bundles are recruited, each with their specific mechanical properties. Hence, the structural properties vary as a function of knee motion. For this reason, a relationship between the structural tensile properties and the relative insertion orientation is required in order to define the role of the ligaments in knee mechanics. In the present study, this relationship is determined by performing a series of tensile tests in which the relative orientations of the insertion sites of human knee bone-ligament-bone preparations were varied systematically. The experimentally obtained stiffness was significantly affected by the relative orientation of the insertion sites, but more profoundly for the anterior and posterior cruciate ligaments (ACL and PCL) as compared to the medial and lateral collateral ligaments (MCL and LCL). The average decreases in stiffness per 5 degrees tilt of the insertion sites were estimated at -11.6 +/- 3.5 N mm-1 (ACL), -20.9 +/- 2.7 N mm-1 (PCL), -2.6 +/- 0.9 N mm-1 (MCL) and -3.7 +/- 0.3 N mm-1 (LCL). For the PCL and the MCL these changes in stiffness with tilt were rather insensitive to the side of the femoral insertion site which was lifted. The ACL and the LCL, conversely, displayed significant differences in stiffness changes between the different tilt directions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗