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At least 811 records · Page 45Linked to original sources
PubMed2001

A new method for detecting pedicular wall perforation during pedicle screw insertion.

STUDY DESIGN: Sensitivity, specificity, and predictive value analyses of a method that can be used to detect pedicular wall perforation during pedicle screw insertion were assessed. OBJECTIVE: To determine the accuracy of observing the fatty material expelled during pedicle screw insertion for detecting pedicular wall perforations. SUMMARY OF BACKGROUND DATA: Although many methods for safe and accurate insertion of a pedicle screw are described, the rates of misplacement still are high, and complications may occur from improper placement of pedicle screws. Computer-assisted insertion techniques are reported to be very accurate, but these techniques are not yet commonly available. METHODS: In this study, 74 pedicle screw insertions were observed for the material expelled after drilling for the pedicle screw. The outflows of blood and fatty particles were recorded separately. The position of the pedicle screws after surgery was verified by computed tomography. The specificity, sensitivity, positive and negative predictive values, and kappa statistics were assessed. RESULTS: Visible fatty particles were observed during 51 pedicle screw insertions. The histologic analysis showed that the fatty particles were coming from the bone marrow of the corpus. For the observation of fatty particles in detecting perforations along the pedicle screw pathway, the specificity was 98%, the sensitivity 73%, the positive predictive value 84%, the negative predictive value 95%, and the kappa statistic 0.74. CONCLUSIONS: The observation of fatty particles in the blood that comes out after drilling for the pedicle screw may indicate that there is no perforation along the pedicle screw pathway. The observation is easy to perform and does not require any special instruments. Therefore, it can be used alone or in combination with other methods to improve the accuracy of pedicle screw insertion.

Adipose Tissue↗
PubMed2001

Does anterolateral cage insertion enhance immediate stabilization of the functional spinal unit? A biomechanical investigation.

STUDY DESIGN: The three-dimensional flexibility of six human lumbar functional spinal units was measured after the anterolateral insertion of an interbody cage. OBJECTIVES: To determine whether an interbody cage inserted from an anterolateral direction stabilizes the spine with respect to the intact state and to compare the finding with that from the same cage inserted from an anterior direction. SUMMARY OF BACKGROUND DATA: Several biomechanical studies have shown that interbody cages do not stabilize the spine in extension. It is suspected that this may be caused by the destruction of the anterior longitudinal ligament and anterior anulus fibrosus. METHODS: Six human cadaveric lumbar functional spinal units were tested under pure moments of flexion, extension, bilateral axial rotation, and bilateral lateral bending to a maximum of 10 Nm. The relative intervertebral motions were measured by an optoelectronic camera system with the spinal units in the intact condition, after discectomy, after anterolateral interbody cage stabilization, and with additional translaminar screw fixation. The implant used was a central, porous, contoured implant with endplate fit. The results were compared with those of a previous study, which used the same implant inserted from an anterior direction. RESULTS: The anterolateral cage insertion significantly decreased the motion in comparison with the intact situation in flexion and lateral bending, but not in extension or axial rotation. No differences were found between the anterior and anterolateral insertion approaches in flexion or extension, but differences were observed in axial rotation and lateral bending, in which the anterolateral approach resulted in more motion. Additional translaminar screw fixation reduced motion to below intact levels in all loading directions. None of the surgical procedures introduced asymmetrical behavior. CONCLUSIONS: Anterolateral cage insertion did not stabilize the spine in extension or axial rotation and was not different from the anterior approach in flexion and extension. Additional translaminar screw fixation stabilized in all directions.

Biomechanical Phenomena↗
PubMed2005

Pullout strength of self-tapping screws inserted to different depths.

OBJECTIVES: The purpose of this study was to determine whether the depth of insertion through the far cortex of self-tapping screws significantly affects pullout strength. DESIGN: Fifty, Synthes, 3.5-mm, self-tapping screws were inserted into synthetic bone blocks and divided into 5 groups. Group 1 had screws with their tips inserted 1 mm short of the far cortex. Group 2 had screws inserted flush with the far cortex. Groups 3, 4, and 5 had screws inserted 1 mm, 2 mm, and 3 mm past the far cortex respectively. Pullout strength was then tested. SETTING: Institutional research laboratory. MAIN OUTCOME MEASUREMENTS: Pullout strength (peak force) was measured for each group and analyzed using a single factor analysis of variance-balanced incomplete block design. RESULTS: Peak force values presented as mean +/- SD for the 5 groups were as follows: group 1 (1380 +/- 69 N), group 2 (1566 +/- 137 N), group 3 (1956 +/- 137 N), group 4 (2013 +/- 184 N), group 5 (2044 +/- 174 N). With a P < or = 0.05, it was found that groups I and II had statistically different pullout strengths than all other groups. However, there was no significant difference in pullout strength between groups 3, 4, and 5. CONCLUSIONS: Synthes self-tapping screws exhibit their highest pullout strength when inserted 1 mm past the far cortex, and there is no significant increase in pullout strength with deeper insertion depths.

Bone Screws↗
PubMed2003

Is there a relationship with anomalous insertions of the distal gubernaculum testis and testicular ectopia? Analysis in human fetuses and patients with cryptorchidism.

PURPOSE: One of the most accepted theories for explaining testicular ectopia is the existence of multiple distal insertions of the gubernaculum testis. We studied the distal insertions of the gubernaculum in human fetuses and patients with cryptorchidism. MATERIALS AND METHODS: We studied 326 gubernacula in 163 human fetuses at 10 to 35 weeks of gestation and 133 gubernacula in 101 patients with cryptorchidism (mean age 6.4 years). With the aid of a stereoscopic microscope (2.5x magnification), the existence of distal insertions of the gubernaculum was investigated. Fisher's exact test was used to compare the 2 study populations. RESULTS: Of 326 fetal testes 224 (68.7%) were abdominal, 45 (13.8%) inguinal and 55 (16.9%) scrotal. In 1 fetus (0.6%) at 23 weeks of gestation both testes and both gubernacula were absent. Among 133 cryptorchid testes 17 (12.8%) were abdominal, 92 (69.2%) inguinal, 24 (18%) high scrotal and 3 (2.3%) evanescent. Of 324 fetal gubernacula present we found only 2 anomalous distal insertions in the pubopenile region (0.6%). All other distal insertions (99.4%) were at the usual location. Of 133 cryptorchid testes the 3 evanescent testes did not present visible gubernacula. Among 130 gubernacula analyzed all were inserted in the scrotal region. CONCLUSIONS: The existence of anomalous insertions at the distal portion of the gubernaculum is rare in human fetuses and patients with cryptorchidism.

Abdomen↗
PubMed1999

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis.

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.

Arabidopsis↗
PubMed1993

Recent stable insertion of mitochondrial DNA into an Arabidopsis polyubiquitin gene by nonhomologous recombination.

Sequence analysis of a newly identified polyubiquitin gene (UBQ13) from the Columbia ecotype of Arabidopsis thaliana revealed that the gene contained a 3.9-kb insertion in the coding region. All subclones of the 3.9-kb insert hybridized to isolated mitochondrial DNA. The insert was found to consist of at least two, possibly three, distinct DNA segments from the mitochondrial genome. A 590-bp region of the insert is nearly identical to the Arabidopsis mitochondrial nad1 gene. UBQ13 restriction fragments in total cellular DNA from ecotypes Ler, No-0, Be-0, WS, and RLD were identified and, with the exception of Be-0, their sizes were equivalent to that predicted from the corresponding ecotype Columbia UBQ13 restriction fragment without the mitochondrial insert. Isolation by polymerase chain reaction and sequence determination of UBQ13 sequences from the other ecotypes showed that all lacked the mitochondrial insert. All ecotypes examined, except Columbia, contain intact open reading frames in the region of the insert, including four ubiquitin codons which Columbia lacks. This indicates that the mitochondrial DNA in UBQ13 in ecotype Columbia is the result of an integration event that occurred after speciation of Arabidopsis rather than a deletion event that occurred in all ecotypes except Columbia. This stable movement of mitochondrial DNA to the nucleus is so recent that there are few nucleotide changes subsequent to the transfer event. This allows for precise analysis of the sequences involved and elucidation of the possible mechanism. The presence of intron sequences in the transferred nucleic acid indicates that DNA was the transfer intermediate. The lack of sequence identity between the integrating sequence and the target site, represented by the other Arabidopsis ecotypes, suggests that integration occurred via nonhomologus recombination. This nuclear/organellar gene transfer event is strikingly similar to the experimentally accessible process of nuclear integration of introduced heterologous DNA.

Amino Acid Sequence↗
PubMed1994

Changes in skin circulation after insertion of a microdialysis probe visualized by laser Doppler perfusion imaging.

Microdialysis makes possible in vivo estimation of endogenous and exogenous substances in the dermal extracellular space. Insertion of the microdialysis probe and its subsequent presence in the skin may affect both the reactivity of the skin test site and the measurement of target substances. Laser Doppler flowmetry is a non-invasive method for estimating cutaneous blood flow. A further development of this technique, laser Doppler perfusion imaging, has been used to study the time course of the circulatory changes caused in the area of microdialysis probe insertion. Laser Doppler perfusion imaging was performed prior to, during, and after microdialysis probe insertion in the skin of the ventral forearm in three subjects. Probe insertion caused an increase in skin blood perfusion in the whole test area. About 15 min after probe insertion, the flare, which is presumed to be of chiefly axon reflex origin, began to subside and the circulatory response could be seen to center around the site of insertion and the tip of the probe. Skin perfusion levels had returned to near normal levels within 60 min. Local anesthesia of the point of guide insertion inhibited the flare, but did not affect circulatory reactivity in the skin nearby. Both microdialysis and laser Doppler perfusion imaging seem to be promising new methods in dermatologic research.

Adult↗
PubMed2004

Peptide insertions in reverse transcriptase pol gene of human immunodeficiency virus type 1 as a rare cause of persistent antiretroviral therapeutic failure.

Peptide insertions in codons 67-71 of the reverse transcriptase (RT) pol gene were detected in 11 (2.7%) of 414 genotypic analyses performed in a hospital cohort of 2900 outpatients with human immunodeficiency virus type 1 (HIV-1) infection. The duration of antiretroviral treatment (bi- or tri-therapy) before the detection of insertions ranged from 12 to 60 months. Dipeptide insertions were detected in ten patients, of which the most frequent was serine-serine. A monopeptide insertion was diagnosed once. The amino-acid composition patterns of insertions varied with time in five of the 11 patients. Peptide insertions were always associated with various patterns of pre-existing or appearing resistance mutations in the RT pol gene to different antiretroviral drugs. Genotypic-guided treatment resulted in virological and immunological improvement in two patients. In contrast, the remaining patients did not respond to any of the various antiretroviral regimens prescribed. Furthermore, various patterns of resistance mutations developed to the prescribed antiretroviral drugs, with AIDS-related conditions leading to death in two patients. It was concluded that peptide insertion in this region of the HIV-1 RT pol gene constitutes a rare cause of persistent therapeutic failure, and that management of such patients remains challenging despite successive genotypic analyses aimed at detecting mutations conferring antiretroviral drug resistance.

Adult↗
PubMed1995

Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the incQ plasmid RSF1010.

Gene cassettes are mobile DNA elements which contain a specific recombination site, a 59-base element, recognized by the site-specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette-associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (Int), and it is likely that the cassette was inserted via a single Int-mediated recombination event between the 59-base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette-associated recombination site was inactivated by the insertion, and Int-mediated excision of the aadB cassette from this non-specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.

Amino Acid Sequence↗
PubMed1994

Effects of amino acid insertions on the proteolysis of a staphylococcal protein A derivative in Escherichia coli.

In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50% in vivo hydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N-terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala-Ile-Ile-Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein.

Amino Acid Sequence↗
PubMed1989

Percutaneous infraclavicular insertion of long-term central venous Hickman catheters.

Percutaneous infraclavicular subclavian vein insertions of single lumen Hickman right atrial catheters (n = 342) were performed on 308 patients at Royal Prince Alfred Hospital. The indications for insertion were administration of total parenteral nutrition (44.8%), intravenous chemotherapy (40.9%), intravenous therapy in patients with inaccessible peripheral veins (11.7%), and intravenous antibiotic administration (2.6%). Three percutaneous catheter insertions were complicated by pneumothorax (0.88%). There were no other complications of insertion. Catheters remained in situ for a median period of 30 days (range: 2-853 days). The majority of catheters (69.6%) remained functioning and complication-free until the completion of therapy or until the patient died of their original disease. Some catheters became infected (9.9%) and there was a 0.6% incidence of septicaemia due to infected catheters; 8.5% of catheters were removed because of a suspicion of infection that was not subsequently proven. The incidence of infection was highest within the first month after catheter insertion, and decreased thereafter. Percutaneous subclavian insertion of Hickman right atrial catheters appears to be the insertion method of choice in patients requiring long-term central venous access.

Catheterization, Central Venous↗
PubMed1992

On the use of multiple probe insertions at the same site for repeated intracerebral microdialysis experiments in the nigrostriatal dopamine system of rats.

The effects of implantation of a dialysis probe into the striatum of awake rats on indices of dopamine (DA) and serotonin neurotransmission were assessed, first over 24 h following initial insertion of a probe, and then again following reinsertion of a probe at the same site 1 week later. It was found that the basal concentration of DA in dialysate stabilized within 20-40 min after probe implantation, although DA showed a modest decline 24 h later. There was, however, no significant difference in basal DA between two test sessions separated by 1 week. On the other hand, the basal concentrations of the DA metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, progressively increased for 2-3 h after probe implantation and decreased markedly by 24 h later. Furthermore, in contrast to DA, the DA metabolites decreased even further after the second probe insertion. Amphetamine-stimulated DA release was also greatly attenuated following the second probe insertion, relative to the first probe insertion. Two probe insertions had only modest effects on the concentration of 5-hydroxyindoleacetic acid in dialysate, relative to the DA metabolites. It is suggested the effects of two probe insertions on DA metabolism and amphetamine-stimulated DA release described here are indicative of probe-induced damage to the nigrostriatal DA system. If this is the case, multiple probe insertions may not provide a feasible strategy for within-subjects design dialysis experiments over extended periods of time, at least in the DA system of small animals. It is suggested further that a stable basal concentration of DA in dialysate may be an especially poor indicator of the integrity of the dopaminergic input to the striatum.

3,4-Dihydroxyphenylacetic Acid↗
PubMedDate not supplied

Pharmacological criteria for ventricular assist device insertion following postcardiotomy shock: experience with the Abiomed BVS system.

BACKGROUND/AIM: The traditional approach to postcardiotomy shock includes inotropic support followed by the application of an intra-aortic balloon pump (IABP). Consideration toward insertion of a ventricular assist device (VAD) becomes necessary when these maneuvers fail to restore hemodynamic stability. The definition of maximal inotropic support, however, is lacking such that a standard formula for VAD insertion remains problematic. The purpose of this paper is to define the pharmacological thresholds for VAD implantation in the setting of postcardiotomy cardiogenic shock. METHODS: The medical records of all adult open-heart operations performed at Hahnemann University Hospital, Philadelphia, PA, from 1 July 1996 through 1 July 1999 were reviewed. Specific data were collected on the hemodynamics and inotrope levels upon separation from cardiopulmonary bypass (CPB). The hospital course was reviewed with attention toward documenting hospital mortality. Cardiogenic shock was defined as systolic blood pressure (SBP) < 100 mmHg, mean pulmonary artery blood pressure (mPAP) > 25 mmHg, central venous pressure (CVP) > 15 mmHg, and cardiac index (CI) < 2.0 L/min/per m2. Inotrope dosages were defined as low, moderate, and high according to assigned values. A formula for VAD insertion was established if cardiogenic shock parameters were present in the setting of two or more high dose inotropes. Early VAD insertion was defined as implantation within three hours of the first attempt to wean from CPB. The VAD recipients were divided into two groups. Group A were VADs placed in conjunction with the formula. Group B was VADs placed in violation (excess) of the formula. The results of these two groups were compared. [table: see text] RESULTS: From 1 July 1996 to 1 July 1999, there were 3462 adult open-heart operations performed at Hahnemann University Hospital, Philadelphia, Pa. The hospital mortality for patients successfully separating from CPB on no inotropes, low-dose, moderate-dose, one high-dose, two high-dose, and three high-dose inotropes were approximately 2.0%, 3.0%, 7.5%, 21%, 42%, and 80% respectively. During this time there were 29 patients supported with the Abiomed BVS (Danvers, Mass) system for postcardiotomy cardiogenic shock. For the entire group of VAD recipients, there were 18 (62%) who were successfully weaned and 8 (28%) who were discharged from the hospital. For the 20 VAD recipients in group A, there were 16 (80%) who were successfully weaned and 8 (40%) who were discharged from the hospital. For the nine VAD recipients in group B, there were two (22%) who were successfully weaned and zero (0%) who were discharged from the hospital. Multiple organ system failure occurred in three (15%) in group A versus seven (78%) in group B patients, respectively. Early VAD insertion was accomplished in 17 (85%) group A patients and 2 (22%) group B patients. CONCLUSIONS: Hospital mortality correlates with the number and level of inotropic support necessary to separate from CPB following adult open heart surgery. The application of a standard pharmacological formula together with hemodynamic criteria for VAD insertion after postcardiotomy cardiogenic shock results in earlier insertion, lower incidence of postoperative MOSF, and improved wean and discharge rates.

Algorithms↗
PubMed1996

Proof of efficacy of different modified sonic scaler inserts used for debridement in furcations--a dummy head trial.

The purpose of this study was to determine the suitability of different scaling instruments for surgical removal of hard and soft bacterial deposits and for the removal of soft accretions only for maintenance treatment within furcations. 12 upper and 12 lower plastic replicated molars, with through-and-through furcations, were instrumented 3 x with 4 different types of instruments: (1) hand instruments; (2) a conventional sonic scaler insert; (3) a set of 3 modified sonic scaler inserts with budded tips and different angulated shafts; (4) a set of 3 sonic scaler inserts with a plastic-coating and different angulated shafts. The plastic replicas were fixed in a dummy head without any replicated soft tissues. In the furcation area, an easily removable surface coating material was applied to the teeth to represent the "plaque" and a second, more stubborn lacquer layer representing "calculus/cementum". Following instrumentation, the following parameters were recorded to assess efficacy; time required for instrumentation, loss of weight, depth of substance removal at the furcation entrance, % of furcation area instrumented, whereby removal of these 2 layers was judged separately. Only minor differences were observed between hand instruments, conventional and budded sonic scaler inserts as to loss of weight, depth of substance loss and area instrumented. The plastic-coated sonic scaler inserts were just as effective in surface layer removal representing "plaque" as the 3 other instruments, but resulted in less loss of weight and less depth of substance removal. In conclusion, the more aggressive hand instruments, the conventional and budded sonic scaler insert, are preferably used for the surgical phase to increased ease of entry into the furcation dome. An effective debridement of the furcation roof seems only possible with an odontoplastic, for which a furcation is fitted to the instrument by means of an intensive instrumentation, thus leading to weight loss and pronounced substance removal. The plastic-coated sonic scaler inserts seems to be a reasonable choice for maintenance treatment within furcation, since this treatment phase is usually restricted to removal of soft bacterial deposits.

Analysis of Variance↗
PubMed2006

Pharmacodynamics of a new ophthalmic mydriatic insert in healthy volunteers: potential alternative as drug delivery system prior to cataract surgery.

Cataract surgery requires a satisfactory degree of mydriasis throughout the entire operation. A phase I, open-labelled, randomised, cross-over trial was conducted in 18 healthy volunteers to compare mydriasis obtained with subsequent administration of phenylephrine 10% and tropicamide 0.5% eyedrops or a new insoluble-matrix retropalpebral ophthalmic insert containing 5.38 mg phenylephrine and 0.28 mg tropicamide. Phenylephrine serum concentrations were measured over 6 hr following each treatment administration. Secondary end-points included cardiovascular, general and local tolerance and quantification of bacterial colonisation of the conjunctiva and the cultured insert, respectively. When normalized to the pupil diameter after conventional treatment, the diameter achieved with the insert was 1.13 (95% confidence interval, 0.94-1.48, P=0.38). Moreover, standard eye drops provided faster effective mydriasis than the insert, starting 30 min. as compared to 90 min. upon treatment administration (P<0.01, repeated-measures ANOVA). Phenylephrine concentrations remained almost undetectable for both treatments and no change in heart rate or blood pressure were observed throughout the study. Only three superficial punctuate keratitis were diagnosed with the insert and two with the eye drops. No significant bacterial contamination of conjunctiva swab and cultured insert was observed. The new insoluble-matrix retropalpebral ophthalmic mydriatic insert produced similar but delayed effective and prolonged mydriasis as compared to the standard delivery system. In addition to its potential usefulness in patients undergoing cataract surgery, such new ophthalmic delivery system may be an advantage in children who need to undergo fundus photography due to the single administration and excellent tolerance as well.

Adolescent↗
PubMed1994

Transposition in Lactobacillus sake and its abolition of lactocin S production by insertion of IS1163, a new member of the IS3 family.

This report presents the nucleotide sequence and insertional activity of IS1163, which is a new member of the IS3 family of transposable elements. Analysis of spontaneous mutants of the lactocin S-producing Lactobacillus sake strain L45 show that the bacteriocin-negative phenotype is due to either loss of the producer plasmid or the insertion of IS1163 into the lactocin S operon (las operon). The data further show that insertional inactivation of the lactocin S operon is the result of a transposition event involving a chromosomally located donor copy of IS1163. Although the insertions described are clustered within a 250-bp region of the las operon, there are no features of the insertion sites to suggest target-specific insertion of IS1163. The overlapping, frameshifted organization of the two major open reading frames found in IS1163 is typical for the IS3 family, but the structure of the putative frameshift region includes features which distinguish IS1163 from the other members of the group. The insertional activity of IS1163 in L. sake L45 has aided in identifying regions of pCIM1 essential for lactocin S production and may have further practical applications as a mutational tool in L. sake.

Amino Acid Sequence↗
PubMed2002

Genetic and phenotypic analysis of Acinetobacter baumannii insertion derivatives generated with a transposome system.

Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.

Acinetobacter baumannii↗
PubMed1991

Expression and immunogenicity of a streptococcal M protein epitope inserted in Salmonella flagellin.

A synthetic 48-bp oligonucleotide specifying the N-terminal 15 amino acids of M protein of Streptococcus pyogenes type 5 (plus a CTA codon, to terminate translation of genes with the insert in reverse orientation) was inserted by blunt-end ligation at the site of the 48-bp EcoRV deletion in the Salmonella flagellin gene in plasmid pLS408 (S. M. C. Newton, C. O. Jacob, and B. A. D. Stocker, Science 244: 70-72, 1989). The resulting plasmid was transferred from Escherichia coli via a restriction-negative Salmonella typhimurium strain into an aromatic-compound-dependent, flagellin-negative live-vaccine strain of Salmonella dublin to produce strain SL7127, which was motile. Expression of the inserted epitope in flagellin and its exposure at the flagellar filament surface were shown by immunoblotting and by the reaction of flagellate bacteria (immobilization, immunogold labeling) with antibody raised by injection of the corresponding synthetic peptide, S-M5(1-15). Rabbits immunized by injection of the live-vaccine strain with flagella composed of the chimeric flagellin or by injection of concentrated flagella from such bacteria developed antibodies reactive in an enzyme-linked immunosorbent assay with peptide S-M5(1-15) and with the large peptic-digest peptide pepM5. These antibodies were opsonic for type 5 streptococci. Mice that were given parenteral live SL7127 (six doses, each 1 x 10(6) to 2 x 10(6), over 8 weeks) developed titers of ca. 12,800 for the M5-specific peptides and opsonizing activity for type 5 streptococci but not for type 24 streptococci. Sera from mice similarly immunized with a control live vaccine strain without an insert in the flagellin gene did not react with the M5-specific antigens. All of the five mice given the control strain, without an insert, died after challenge with type 5 streptococci or type 24 streptococci; by contrast, four of the five mice given strain SL7127, with an insert, survived the M5 challenge, but none of the five challenged with the type 24 strain survived. Therefore, our study shows that an M protein epitope can be expressed in the context of an unrelated protein and maintain its immunogenicity. Furthermore, we demonstrate that mice can be protected against a Streptococcus pyogenes type 5 challenge by immunization with a Salmonella live vaccine with flagella made of flagellin with an insert carrying a protective epitope of M5 protein but without the cross-reactive epitopes of the complete protein.

Amino Acid Sequence↗