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At least 505 records · Page 28Linked to original sources
PubMed1996

A comparative study of two techniques used in immediate postplacental insertion (IPPI) of the Copper T-380A IUD in Shanghai, People's Republic of China.

Chinese women in Shanghai who delivered vaginally and who chose to use an IUD for contraception received a Copper T-380A IUD inserted vaginally within 10 minutes after delivery of the placenta (i.e., immediate postplacental insertion, IPPI). Among them, 97.7% were primipara. The women were randomly divided into two groups: IUD inserted by hand and IUD via ring forceps. The follow-up rate of six months was 95.2%. Using Tietze's life table method and log rank test, the expulsion and other discontinuation rates were compared at three and six months postinsertion between these two different insertion techniques. Expulsions were the main reason for discontinuation. The six-month gross cumulative expulsion rates were 13.3 and 12.7 per 100 women in the hand-insertion group and ring forceps-insertion group, respectively. Discontinuation rates for medical removals (bleeding/pain) were 2.1 and 1.0 in these two groups, respectively. Neither of the differences was statistically significant (p > 0.05). No uterine perforation, infection or pregnancy occurred. The results suggest that these two different insertion techniques do not significantly affect discontinuation rates in vaginal IPPI using the TCu 380A, and the TCu 380A appears to be suitable for postpartum insertion in Chinese women. Other relevant issues, such as breastfeeding and IUD placement in uterine cavity, are also analyzed and discussed in this report.

Adult↗
PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗
PubMed2003

Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP.

The Escherichia coli GABA (gamma-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [(3)H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [(3)H]nipecotic acid and [(14)C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in k (cat)/ K (m)). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [(3)H]nipecotic acid relative to [(14)C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842-5847] that have a functionally significant CAR-like domain.

Alanine↗
PubMed2006

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

DNA Transposable Elements↗
PubMed1995

Naturally occurring tyrosine kinase inserts block high affinity binding of phospholipase C gamma and Shc to TrkC and neurotrophin-3 signaling.

Neurotrophin-3 binds to the receptor tyrosine kinase, TrkC. Several naturally occurring splice variants of TrkC exist including those with 14- and 39-amino acid inserts within the tyrosine kinase homology region. When expressed in fibroblasts, full-length TrkC, but not the kinase insert variants, mediated neurotrophin-3-stimulated cell proliferation. We investigated the molecular basis of this signaling defect. The kinase inserts blocked the ability of TrkC to mediate neurotrophin-3 stimulated c-myc and c-fos transcription and activation of the AP-1 transcriptional complex. In cells expressing full-length TrkC, neurotrophin-3 promoted a sustained activation of mitogen-activated protein kinase; TrkC containing kinase inserts only mediated transient activation of mitogen-activated protein kinase. The kinase inserts specifically blocked neurotrophin-3-stimulated autophosphorylation of the phospholipase C gamma binding site on TrkC (tyrosine 789) resulting in a severe reduction in phospholipase C gamma association with TrkC and its tyrosine phosphorylation. Neurotrophin-3-stimulated phosphorylation of the Shc binding site (tyrosine 485) on TrkC, and tyrosine phosphorylation of Shc itself, was unaffected by the kinase inserts; however, the kinase inserts blocked high affinity Shc association with TrkC. It is proposed that the lack of high affinity binding of Shc and/or phospholipase C gamma to the TrkC kinase insert variants may be responsible for the inability of these variants to bring about a full biological response in fibroblasts.

3T3 Cells↗
PubMed2006

Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae.

Transposons are widely employed as tools for gene disruption. Ideally, they should display unbiased insertion behavior, and incorporate readily into any genomic DNA to which they are exposed. However, many transposons preferentially insert at specific nucleotide sequences. It is unclear to what extent such bias affects their usefulness as mutagenesis tools. Here, we examine insertion site specificity and global insertion behavior of two mini-transposons previously used for large-scale gene disruption in Saccharomyces cerevisiae: Tn3 and Tn7. Using an expanded set of insertion data, we confirm that Tn3 displays marked preference for the AT-rich 5 bp consensus site TA[A/T]TA, whereas Tn7 displays negligible target site preference. On a genome level, both transposons display marked non-uniform insertion behavior: certain sites are targeted far more often than expected, and both distributions depart drastically from Poisson. Thus, to compare their insertion behavior on a genome level, we developed a windowed Kolmogorov-Smirnov (K-S) test to analyze transposon insertion distributions in sequence windows of various sizes. We find that when scored in large windows (>300 bp), both Tn3 and Tn7 distributions appear uniform, whereas in smaller windows, Tn7 appears uniform while Tn3 does not. Thus, both transposons are effective tools for gene disruption, but Tn7 does so with less duplication and a more uniform distribution, better approximating the behavior of the ideal transposon.

Base Sequence↗
PubMed1999

A large-scale insertional mutagenesis screen in zebrafish.

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Alleles↗
PubMed1994

The non-random pattern of insertion of IS2 into the hemB gene of Escherichia coli.

The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2. The insertional specificity of IS2 is still unclear. This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity. The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5' end of the gene (region I). Twenty-one of 24 insertions occurred in region I. Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3' terminal segment. A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3' terminus has been identified in region I. Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites.

Bacterial Proteins↗
PubMed2004

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Acetyltransferases↗
PubMed2001

Insertion-duplication mutagenesis of neisseria: use in characterization of DNA transfer genes in the gonococcal genetic island.

We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

Chromosomes, Bacterial↗
PubMed1999

Sequence and insertion sites of murine melanoma-associated retrovirus.

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and pol regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3' coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-maf proto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3' noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

Animals↗
PubMed2001

Retrovirus insertion and transcriptional activation of the multidrug-resistance gene in leukemias treated by a chemotherapeutic agent in vivo.

To understand the molecular basis for multidrug-resistant (MDR) cancer cells in vivo, this study analyzed molecular changes of the mdr1a gene region in leukemia cells in mice during continuous treatment with vincristine. An inverse insertion of murine leukemia retrovirus (MuLV) into the 5'-flanking region of the mdr1a gene was found. This insertion was concomitantly accompanied by up-regulation of the mdr1a gene and the loss of chemosensitivity. Deletion of long-terminal repeat (LTR) sequences dramatically decreased the mdr1a promoter-driven reporter activity. The MuLV LTR insertion appears to exert its enhancer activity on mdr1a transcription during the appearance of MDR leukemia cells. Two mechanisms were postulated to explain the mdr1a gene activation by retrovirus insertion during in vivo chemotreatment: de novo insertion of MuLV induced by vincristine treatment and selection of a small fraction of pre-existing cells carrying MuLV insertion during vincristine treatment. No rearranged sequence was detected by polymerase chain reaction in parental cells. This result argued for the first mechanism. The randomly altered distribution of MuLV during repetitive chemotreatment might also be consistent with this hypothesis. On the other hand, the retrovirus insertion was detected at the same site of the mdr1a promoter region in 2 independent experiments, which suggests the second mechanism. It should be noted that in vivo chemotreatment using vincristine could generate the mdr1a-overexpressing cells through retrovirus insertion and the enhancer effect of the LTR.

ATP Binding Cassette Transporter, Subfamily B↗
PubMedDate not supplied

Identification of insertion mutations in HIV-1 reverse transcriptase causing multiple drug resistance to nucleoside analogue reverse transcriptase inhibitors.

OBJECTIVE: A novel 2-amino acid insertion between codons 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) which confers multiple drug resistance has recently been reported. Independently, we have identified similar insertion mutations in Japanese hemophiliacs and attempted to analyze their emergence in conjunction with therapy regimens and their contribution to drug resistance using recombinant technology. METHODS: The plasma and peripheral blood mononuclear cells (PBMCs) of 348 HIV-1-infected hemophiliacs were screened for HIV-1 RT mutations relevant to nucleoside analogue inhibitors and isolating viruses. Contribution of each insertion to drug resistance was studied by introducing the mutations into a T-cell line-tropic NL4-3 infectious clone and testing the drug susceptibilities of the recovered virus. RESULTS: Insertion of the 2-amino acid residue was found in 4 of the 348 cases and was strongly associated with prolonged chemotherapy with zidovudine (AZT) and didanosine (ddI). The virus isolated from 1 of the 4 cases possessed the same insertion. Characterization of these virus and the recombinant NL4-3 with the insertion strongly suggested that the insertion caused resistance not only to AZT and ddI but also to lamivudine (3TC) and zalcitabine (ddC). CONCLUSION: A 2-amino acid insertion between codons 69 and 70 of RT was detected in 4 of 348 (1.1%) Japanese hemophiliacs and was found to be associated with multiple drug resistance to nucleoside analogue RT inhibitors.

Amino Acid Sequence↗
PubMed1983

Failure to insert an intrauterine device.

Numerous studies have reported IUD-related events in evaluating the rates of pregnancy, expulsion, and removal for bleeding and pain, but the inability to successfully insert an IUD has been overlooked. This study analyzed data on 51 unsuccessful insertions from 22 international centers collected on standard forms. The incidence of insertion failures was estimated to be between 2.3 and 8.3/1000 attempted insertions. No statistically significant risk factors in patient characteristics were delineated through a case-control analysis. The incidence of insertion failures among postpartum cases was highest for the Delta Loop (16.8/1000 insertions). Data suggest that failure to successfully insert an IUD may not be directly attributable to any specific host characteristic but rather is contingent on agent (IUD type) and provider (inserter) factors.

Cervix Uteri↗
PubMed1994

Single amino acid insertions probe the alpha subunit of the Escherichia coli F1F0-ATP synthase.

Single amino acid insertions of alanine or aspartate have been introduced into the alpha subunit of the F1F0-ATP synthase at seven different sites, after residues 187, 193, 198, 202, 212, 217, and 222. These sites span a highly conserved region of the alpha subunit, parts of which are thought to be located in transmembrane spanning regions. Alanine insertions have little or no effect on function after positions 187, 193, 198, and 202, indicating that the region spanned by these residues is not essential for function. Alanine insertions after residues 212 and 217 disrupt ATP synthase function without grossly affecting the assembly of the enzyme, while the alanine insertion after residue 222 disrupts both ATP synthase function and assembly. All of the aspartate insertions are deleterious to ATP synthase function, except after residue 198. At the other six sites, aspartate insertions prevent growth on succinate minimal medium, indicating an inability to synthesize ATP. Aspartate insertions after residues 187 and 193 result in alpha subunits that do not fractionate with membranes, as indicated by immunoblotting. These results support a model of the alpha subunit in which residues 187-193 and residues 212-222 are part of distinct transmembrane spans, separated by a short extramembrane loop. The results are consistent with an important interaction between residues 212-222 of the alpha subunit and b or c subunits. General aspects of "insertion scanning mutagenesis" are also discussed.

Amino Acid Sequence↗
PubMed1989

The relationship between intraoperatively assessed atrial and ventricular insertions of accessory pathways.

To gain insight into the complex problems regarding the extension of the atrial and ventricular insertions of anomalous pathways, the activation patterns obtained from intraoperative epicardial mapping in 23 patients with Wolff-Parkinson-White syndrome were carefully analyzed. The atrial and ventricular activation times along the atrioventricular groove were measured at 15 predefined anatomical landmarks. The width of the region of earliest atrial and ventricular activation times and the extent of atrial and ventricular overlap were assessed. The relationship between the atrial and ventricular insertions was studied by predicting the ventricular insertions from the observed atrial insertions and vice versa. The mean extension of an atrial and a ventricular insertion at surgery was 1.9 +/- 0.2 (SE) and 1.6 +/- 0.2 (SE) anatomical landmarks, respectively. The width of the region of early atrial and ventricular activation times measured 2.7 +/- 0.3 (SE) landmarks, thus indicating an overlap of the atrial and ventricular insertions. The lateral distance between the sites of earliest atrial and ventricular activation times was, on average, 1.7 +/- 0.3 (SE) landmarks. The predicted atrial insertion from a known ventricular insertion, and vice versa was found to cover 4 to 6 anatomical landmarks. These observations suggest that wide surgical dissections along the atrioventricular groove are warranted even if epicardial mapping discloses only a single accessory pathway.

Action Potentials↗
PubMed2001

Effects of in vitro wear of machined and molded UHMWPE tibial inserts on TKR kinematics.

The effect of manufacturing process on the wear and mechanical performance of a total knee replacement (TKR) design was investigated with the use of a force-controlled knee joint simulator. Ultra-high molecular weight polyethylene (UHMWPE) tibial inserts processed by direct compression molding from 1900H resin were compared to UHMWPE tibial inserts machined from a compression-molded sheet of GUR 1050. Both sets of components had the same posterior-cruciate-retaining geometry, and were identically aligned with cobalt-chromium-molybdenum alloy femoral components. Wear tests were conducted at a frequency of 1 Hz for 4 million cycles with the use of a standard walking cycle pattern. Implant kinematics, including anterior-posterior (AP) displacement and internal-external (IE) rotation in response to applied loads were monitored. Gravimetric wear, surface roughness, and surface morphology were used to characterize the wear process of the UHMWPE inserts. Results showed that the molded UHMWPE inserts exhibited less gravimetric wear over time than the machined inserts of the same design. Both the machined and molded components exhibited scratching, pitting, and burnishing over their wear areas. The AP displacement distance per cycle of the molded tibial inserts decreased over the course of testing, resulting in a shorter total testing displacement for this group compared to machined tibial inserts. Although AP displacement distance per cycle for machined tibial inserts did not change significantly over the course of testing, their position relative to the femoral components shifted posteriorly over time, resulting in an elongated wear track.

Arthroplasty, Replacement, Knee↗
PubMed2006

Propagated insertional activity: a model of positive sharp wave generation.

In this study we utilized a dual monopolar needle recording technique to assess propagated electromyographic insertional activity from the same single muscle fiber in order to characterize different categories of insertional activity. A total of six combinations of insertional activity were identified. Only two fundamental types of single muscle-fiber insertional discharge configurations were generated: biphasic initially-negative and monophasic positive. The propagated waveforms corresponding to these two insertional discharges were primarily triphasic initially-positive and, only rarely, monophasic positive. The monophasic positive insertional activity generated at the inserting electrode site is postulated to arise from a depolarization zone adjacent to a needle-induced peri-electrode membrane crush. The monophasic positive discharge was utilized as a model for positive sharp wave generation. It is postulated that the majority of positive sharp waves are initiated at the inserting electrode adjacent to a needle-induced zone of muscle membrane crush in contrast to the previous supposition that positive sharp waves are blocked fibrillation potentials.

Action Potentials↗