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PubMed1997

Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase.

The TEM-1 beta-lactamase enzyme efficiently hydrolyzes beta-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant beta-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of beta-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant beta-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Omega-loop region, which forms part of the catalytic pocket of the beta-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Omega-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Omega-loop with regards the primary structure requirements for ceftazidime hydrolysis by beta-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed.

Amino Acid Sequence↗

PubMed2001

Genomic characterization of recent human LINE-1 insertions: evidence supporting random insertion.

LINE-1 (L1) elements play an important creative role in genomic evolution by distributing both L1 and non-L1 DNA in a process called retrotransposition. A large percentage of the human genome consists of DNA that has been dispersed by the L1 transposition machinery. L1 elements are not randomly distributed in genomic DNA but are concentrated in regions with lower GC content. In an effort to understand the consequences of L1 insertions, we have begun an investigation of their genomic characteristics and the changes that occur to them over time. We compare human L1 insertions that were created either during recent human evolution or during the primate radiation. We report that L1 insertions are an important source for the creation of new microsatellites. We provide evidence that L1 first strand cDNA synthesis can occur from an internal priming event. We note that in contrast to older L1 insertions, recent L1s are distributed randomly in genomic DNA, and the shift in the L1 genomic distribution occurs relatively rapidly. Taken together, our data indicate that strong forces act on newly inserted L1 retrotransposons to alter their structure and distribution.

3' Flanking Region↗

PubMed2003

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome.

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.

Base Composition↗

PubMed1985

Comparison of intramenstrual IUD insertion with insertion following menstrual regulation.

To evaluate the use-effectiveness and safety of IUD insertion immediately after menstrual regulation (MR) for delayed menses, a Lippes Loop D (LLD) intrauterine device was inserted in each of 100 consecutive clients at the University College Hospital, Ibadan, immediately after menstrual regulation. Pertinent event rates after 12 months of use were compared with those of 100 consecutive women who had the LLD inserted during menstruation. The cumulative net expulsion rate after 12 months of use was 8% for the study group and 4% for the controls. The overall rate of removals was 15% for the study group and 16% for the control group. None of the observed differences was statistically significant. The continuation rates at 12 months were comparable for both groups (78% and 80%, respectively). There were no accidental pregnancies during the study period. The authors suggest that IUD insertion immediately after menstrual regulation is as effective and safe as intramenstrual insertion, provided prophylactic antibiotics are given.

Adult↗

PubMed1997

Autodirected insertion: preinserted VDAC channels greatly shorten the delay to the insertion of new channels.

VDAC, a mitochondrial outer membrane channel, has the ability to catalyze and direct the insertion of other VDAC channels into planar phospholipid membranes. The spontaneous rate of insertion of detergent-solubilized VDAC channels into phospholipid membranes is estimated to be 1.5 x 10(-5) channels min-1 micron-2. VDAC channels already in the membrane can increase this rate by a factor of 10(9). The presence of 5 M urea on the opposite side of the membrane increases this 10-fold to 4.5 x 10(5) channels min-1 microns-2. Similar but weaker effects are observed with Triton X100 addition (10(-3)% (v/v)). These agents are not acting on uninserted channels because they do not affect the delay from sample addition to first insertion. Under the chosen conditions, this delay is long (240 s) without preinserted channels. However, the presence of a few VDAC channels in the membrane reduces this delay to 14 s, close to the diffusion limit. Therefore, urea and Triton, added to the side of the membrane opposite that to which the VDAC sample was added, likely increase the flexibility of the VDAC channels in the membrane, allowing them to be more efficient catalysts for VDAC insertion. There are obvious implications for membrane protein insertion and targeting.

Cell Membrane↗

PubMed1998

Establishing an ultrasound guided peripherally inserted central catheter (PICC) insertion service.

Peripherally inserted central catheters (PICCs) are an increasingly used means of medium to long-term intravenous access. We have established an ultrasound (US) guided PICC insertion service at our institution that has largely replaced blind bedside insertions. One hundred and six PICCs were inserted in the upper arms of 89 patients, over a 6-month period with a 100% success rate. The technique for US guided vein puncture and guidewire insertion is described. Catheter related complications included infection (6 proven cases; 3.1 infections per 1000 catheter days); catheter blockage (7), breakage (8) and unintentional removal (5). Catheter blockage, breakage and partial unintentional removal are potentially remediable complications using guide-wire techniques under fluoroscopic guidance. The management of catheter related infection is discussed. We have encouraged early communication with our clinical colleagues in order to facilitate the management of such complications and minimize unnecessary catheter removal.

Catheterization, Central Venous↗

PubMed2002

Mechanism of propylene insertion using bis(phenoxyimine)-based titanium catalysts: an unusual secondary insertion of propylene in a group IV catalyst system.

A highly regioselective secondary enchainment of propylene in a group IV catalyst system is reported. End-group analysis of polypropylene formed using the phenoxyimine-based titanium catalysts revealed a reversal in the regioselectivity of insertion for this class of catalysts. To the best of our knowledge, bis(phenoxyimine)-based titanium complexes are the only known group IV catalysts that insert propylene with exclusive 2,1-regiochemistry. Insertion of propylene into the initiating titanium hydride occurs with high 1,2-regiochemistry. Subsequent insertions into primary titanium alkyls are regiorandom, while insertions into secondary titanium alkyls proceed with high 2,1-regioselectivity. Cyclopolymerization and ethylene/propylene copolymerization strategies are employed to support this proposal.

Alkenes↗

PubMed1998

The interface between bone and tendon at an insertion site: a study of the quadriceps tendon insertion.

Traumatic avulsions of ligament or tendon insertions rarely occur at the actual interface with bone, which suggests that this attachment is strong or otherwise protected from injury by the structure of the insertion complex. In this study we describe the terminal extent of quadriceps tendon fibres where they insert into the patellae of adult rabbits, humans, dogs and sheep. Specimens were examined by scanning electron microscopy (SEM) and light microscopy (LM). To facilitate tracing of tendon fibres the specimens were decalcified for SEM, and polarised light microscopy (PLM) was used in the LM segment of the study. By SEM it was possible to identify mature bone by the presence of osteocytes and a lamellar organisation. PLM and SEM showed that, unlike tendon fibres elsewhere, those in the calcified fibrocartilage were not crimped. No specific cement line was identified by SEM. Tendon fibres interdigitated among separate bone lamellar systems, (osteons or marrow spaces), but did not merge with the collagen systems of individual lamellae. The interdigitation was more extensive and the margin between tendon and bone was less distinct in the anterior third of the insertion. The segment of calcified tendon which interdigitated with bone stained less intensely blue and was less cellular than the more proximal calcified fibrocartilage zone adjacent to the tidemark. Lamellar collagen fibres of the bony trabeculae in the anterior patella were unusually parallel and longitudinal in orientation, making distinction of interposed tendon fibres difficult on LM and PLM sections. LM, SEM and transmission electron microscopy of rabbit patellae at birth revealed that anterior quadriceps tendon fibres extended over the patella in a fibrous cellular layer. By 2 wk of age, this layer had acquired chondroid features (i.e. cell lacunae and metachromasia) and contained vessels extending from patellar marrow. At 6 wk of age, part of this fibrocartilaginous layer was replaced by mature bone and osteoid. In the young adult animal, the quadriceps tension interdigitates extensively with the patellar bone. This segment of the insertion is perhaps the remnant of calcified fibrocartilage which has been remodelled by bone formation.

Aged↗

PubMed1982

Cell surface insertion of exogenous epidermal growth factor receptors into receptor- mutant cells: demonstration of insertion in the absence of added fusogenic agents.

We show that epidermal growth factor (EGF) receptor can be transferred in a biologically active orientation from donor hepatic membranes to recipient receptorless fibroblast cells. The recipient cells (NR-6) normally lack EGF receptors and are biologically unresponsive to EGF. The transfer of receptors from donor plasma membranes to recipient NR-6 surface membranes occurs in the absence of any added fusogenic agent. Studies on time and temperature dependence of this transfer indicate that it is due to preferential insertion of the EGF receptor over the other hepatic proteins. The inserted receptor is exceptionally stable to dissociation or damage, and this facilitated studies on its biological properties. The inserted receptor confers upon the hitherto unresponsive variant NR-6 cells a specific biological responsiveness to EGF as measured by EGF-induced stimulation of DNA replication and cell division. These findings suggest the existence of an affinity-mediated mechanism for the biologically active insertion of exogenous EGF receptors into receptorless variant cells. This insertion approach may be of use in the identification of receptor-associated membrane proteins that play a role in the transmission of EGF biological message.

Animals↗

PubMed1999

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2.

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.

3T3 Cells↗

PubMedDate not supplied

Enhancing patient safety during feeding-tube insertion: a review of more than 2,000 insertions.

BACKGROUND: An intervention to reduce complications from insertion of small-bore nasogastric feeding tubes was performed. METHODS: This was a Performance Improvement project with the Plan, Do, Study, Act (PDSA) format; interventions occurred in July 2003. Electronic searches of risk management and radiology databases identified feeding-tube malpositions and complications from January 1, 2001, through December 31, 2004. Chart abstraction and a pre- and postintervention comparison were performed. Interventions were adoption of a more compliant feeding tube, direct supervision of residents, technology-guided insertion, and implementation of explicit policies and procedures. RESULTS: Of all small-bore nasogastric feeding-tube placements, 1.3%-2.4% resulted in 50 documented cases of feeding-tube malpositions during 4 years. Over half of the 50 patients were mechanically ventilated, and only 2 had a normal mental status. There were 13 complications (26% of malpositions), including 2 deaths, which were directly attributed to the feeding-tube malposition. Only 2 of the 13 complications and none of the misplacements had been recorded in the risk management database; most cases were identified from the search of radiology reports. In the 15-month postintervention period, no complications were identified. The control chart showed that after the intervention, there was a significant increase in the "number between" tube insertions without complications, confirming the effectiveness of the performance improvement (PI) project. CONCLUSIONS: Unassisted feeding tube insertion carries significant risk in vulnerable patients, which can be mitigated. Voluntary reporting appears inadequate to capture complications from feeding tube insertion.

Enteral Nutrition↗

PubMed2003

Membrane insertion of the chloroplast outer envelope protein, Toc34: constrains for insertion and topology.

The insertion of the outer envelope protein Toc34 from chloroplasts was studied. Toc34 was chosen as a model protein because it contains one predicted transmembrane helix at the C-terminus and a large hydrophilic N-terminal located GTPase domain, which is exposed to the cytosol. Unlike proteins located in internal chloroplast compartments, Toc34 neither contains a cleavable presequence nor uses the general import pathway. The protein can insert into the outer envelope of chloroplasts but not into the outer membrane of mitochondria. Using protein-free liposomes we showed that Toc34 is able to insert directly into the lipid bilayer. This insertion is stimulated by GTP and the presence of nonbilayer lipids, but is independent of the presence or absence of charged lipids. The topology of the protein inserted into protein-free liposomes was not exclusively directed by the positive-inside rule but by the size of the hydrophilic domain.

Adenosine Triphosphate↗

PubMed1997

Cochlear implant deep electrode insertion: extent of insertional trauma.

We have recently undertaken deep insertions of the Combi-40 cochlear implant electrode (Med-E1 Corp., Innsbruck, Austria) into apical regions of the scala tympani using a cochleostomy approach. In order to examine the extent of the insertional trauma, 12 fresh human temporal bones were implanted with original Combi-40 electrodes. The specimens were histologically processed with the implants in place by employing a sawing and grinding technique. In most cases, only very discrete distortions of the epithelium of the spiral ligament occurred within the middle cochlear turns. Furthermore, a slight displacement of the basilar membrane caused by the electrode was occasionally seen. However, in 2 cases more severe damage such as basilar membrane rupture and electrode displacement was found. Attempts to insert the electrode beyond the point of first resistance resulted in electrode kinking within the basal cochlear turn with subsequent fracture of the osseous spiral lamina. According to our results, deep electrode insertions do not aggravate the insertional trauma provided no force is applied when resistance is felt.

Cochlea↗

PubMed1999

[Comparison between copper needle insertion and stainless steel needle insertion into rabbit ear central veins].

OBJECTIVE: The clinical application of copper needle insertion has been proceeded satisfactorily for peripheral vascular disorders. This work was to study the mechanism of the copper needle insertion on a pathological basis. METHODS: Sixty rabbits were used in the experiments. The copper needle or the stainless steel needle was inserted into the ear central vein of the animal. Then the specimens of the central vein including its surrounding tissue were taken for light and electron microscopic examinations. RESULTS: At 7 days after copper needle insertion, acute inflammation reaction of the venous wall and vascular thrombosis were revealed in the specimens. At 14 days, giant cells granuloma and destruction of the venous wall were seen. After 28 days to 2 months, granuloma and the residual destroyed venous wall were gradually absorbed and fibrosis resulted. The vascular inflammation and thrombosis caused by stainless steel needle insertion was slight and slow. CONCLUSION: Copper needle treatment for vascular disorders is superior to the stainless steel needle.

Animals↗

PubMedDate not supplied

Peripherally inserted central catheter insertion problems associated with topical anesthesia.

Application of the topical anesthetic cream to the antecubital fossa 60 minutes before insertion of the peripherally inserted central catheters resulted in significant vasoconstriction and/or vasospasm in 24 patients. Catheter insertion was difficult in all 24 patients. Advancement of the peripherally inserted central catheter was impossible in six patients. Of the 51 patients that did not have the topical anesthetic pre-catheter insertion, there were no difficulties in placing the lines, nor were there any vasoconstriction and/or vasospasm. This group included 66 adults and 9 children.

Aged↗

PubMed2004

Nogo 3'-untranslated region CAA insertion: failure to replicate association with schizophrenia and demonstration of marked population difference in frequency of the insertion.

Novak et al. [Brain Res. Mol. Brain Res. 107 (2002) 183] reported that a CAA insertion in the 3'-untranslated region of the Nogo gene was associated with schizophrenia. We examined the frequency of this CAA insertion in 57 European American subjects with schizophrenia and 243 controls, and in a smaller group of African American subjects (N=72; 20 with schizophrenia). We found a similar frequency of the CAA insertion for patients and controls in both populations, but a large difference in CAA insertion frequency between the two racial groups.

Black or African American↗

PubMed2006

Analysis of T-DNA insertion site distribution patterns in Arabidopsis thaliana reveals special features of genes without insertions.

Large collections of sequence-indexed T-DNA insertion mutants are invaluable resources for plant functional genomics. Flanking sequence tag (FST) data from these collections indicated that T-DNA insertions are not randomly distributed in the Arabidopsis thaliana genome and that there are still a fairly high number of annotated genes without T-DNA insertions. We have analyzed FST data from the FLAGdb, GABI-Kat, and SIGnAL mutant populations. The lack of detectable transcriptional activity and the absence of suitable restriction sites were among the reasons genes are not covered by insertions. Additionally, a refined analysis of FSTs to genes with annotated noncoding regions showed that transcription initiation and polyadenylation site regions of genes are favored targets for T-DNA integration. These findings have implications for the use of T-DNA in saturation mutagenesis and for our chances to find a useful knockout allele for every gene.

Arabidopsis↗

PubMed1999

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions.

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.

Chloramphenicol Resistance↗