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PubMed1999

Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition.

The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse-chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.

Endo-1,4-beta Xylanases↗
PubMed1995

Comparison of endolymph cross-sectional area measured histologically with that measured in vivo with an ionic volume marker.

In order to establish how endolymph volume is regulated, it is essential to be able to measure endolymph volume or cross-sectional area in vivo. We have developed methods to accomplish this by injecting the volume marker ion hexafluoroarsenate (AsF6) into endolymph by iontophoresis. For an injection at a constant rate, the endolymph concentration is inversely dependent on the cross-sectional area of the scala into which injection occurred. Marker concentrations were monitored by inserting ion-selective microelectrodes into endolymph near the injection site. In a previous study we quantified the degree of hydrops in animals following ablation of the endolymphatic sac. In the present study we validated the technique by comparing the endolymphatic cross-sectional area measured in vivo with AsF6 with that measured by established histologic procedures. The correlation between the two measures was good, with a coefficient of .903, although the area measured histologically was a little lower than that measured in vivo.

Animals↗
PubMedDate not supplied

Inverse intubation in air medical transport.

INTRODUCTION: The purpose of this study was to investigate the speed and accuracy of the inverse intubation procedure. TECHNIQUE: The operator crouches or kneels over the patient, straddling the torso. The laryngoscope is held in the operator's right hand in an overhand fashion, inserted gently into the patient's mouth, and pulled up and caudad. When the vocal cords are visualized, the endotracheal tube is passed with the left hand. METHODS: The procedure was taught to flight nurses and respiratory therapists (n = 21) using an intubating mannequin strapped to a stretcher in a BK 117 helicopter to simulate an in-flight intubation. The subjects were timed intubating the mannequin using both inverse and standard intubation techniques. Each technique was performed twice, and the times were averaged. The Wilcoxon Signed Ranks test was used to determine statistical significance. RESULTS: No significant difference occurred between times of the standard (24.0 s) and inverse techniques (21.6 s) (P =.715) or number of attempts for successful endotracheal intubation (1.12 and 1.07, P =.581). CONCLUSION: Inverse intubation is a useful skill for prehospital providers. This skill can be taught in a brief period and used successfully with no compromise in speed or success rate.

Air Ambulances↗
PubMed1976

[Pericentric inversion of chromosome 3, homozygote and heterozygote, and centromeric translation of chromosome 12 in a family of orangutangs. Evolutionary implications].

An orangutang family is reported in which the father (Piku) is homozygous for a pericentric inversion of chromosome 3 and heterozygous for a structural rearrangement of chromosome 12 (author's nomenclature for this species). The latter is interpreted as transposition of the centromere by insertion into band q213 [in cen (12)(q213)]. Piku's mate has a normal female chromosome complement. His daughters by this mate (Agnès and Ursula) are heterozygous for both his rearrangements.

Animals↗
PubMed2006

Modifying yeast artificial chromosomes to generate Cre/LoxP and FLP/FRT site-specific deletions and inversions.

The ability to efficiently and accurately modify genomic DNA through targeted and tissue-specific mutations is an important goal in animal transgenesis. Here we describe how to exploit two systems of homologous recombination, from yeast and bacteria, to engineer yeast artificial chromosomes (YACs) to generate targeted deletions and inversions in vivo, in transgenic animals, and in the presence of DNA-modifying enzymes known as recombinases. Through homologous recombination in yeast, specific recombinogenic sequences are inserted upstream and downstream of a region in the YAC. The sites of integration of these short sequence elements are chosen carefully, such that the YAC is left functionally intact, and this modified transgene represents the wild-type allele. This YAC is subsequently used to generate transgenic animals, which when bred to animals expressing recombinase proteins result in genetic modifications. By expressing recombinase proteins from different tissue-specific promoters, one can mediate site-specific recombination to generate either ubiquitous or tissue-specific deletions or inversion. These modifications can then be carried through the germline or can be studied somatically. A great advantage of this system is the ability to evaluate subtle genetic effects independent of position-effect variegation, and transgene copy number, eliminating the need to examine several independently generated lines of transgenic animals for each genetic variant.

Animals↗
PubMed1997

Characterization of ICP6::lacZ insertion mutants of the UL15 gene of herpes simplex virus type 1 reveals the translation of two proteins.

The herpes simplex virus type 1 (HSV-1) UL15 gene is a spliced gene composed of two exons and is predicted to encode an 81-kDa protein of 735 amino acids (aa). Two UL15 gene products with molecular masses of 75 and 35 kDa have been observed (J. Baines, A. Poon, J. Rovnak, and B. Roizman, J. Virol. 68:8118-8124, 1994); however, it is not clear whether the smaller form represents a proteolytic cleavage product of the larger form or whether it is separately translated. In addition, an HSV-1 temperature-sensitive mutant in the UL15 gene (ts66.4) is defective in both cleavage of viral DNA concatemers into unit-length monomers and packaging of viral DNA into capsids (A. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993; J. Baines et al., J. Virol. 68:8118-8124, 1994). In this study, we detected two UL15 gene products of 81 and 30 kDa in HSV-1-infected cells, using a polyclonal antibody raised against a maltose binding protein fusion construct containing UL15 exon 2. In addition, we report the isolation of two HSV-1 insertion mutants, hr81-1 and hr81-2, which contain an ICP6::lacZ insertion in UL15 exon 1 and exon 2 and thus would be predicted to encode C-terminally truncated peptides of 153 and 509 aa long, respectively. hr81-1 and hr81-2 are defective in DNA cleavage and packaging and accumulate only B capsids. However, both mutants are able to undergo wild-type levels of DNA replication and genomic inversion, suggesting that genomic inversion is a result of DNA replication rather than of DNA cleavage and packaging. We also provide evidence that the 81- and 30-kDa proteins are the products of separate in-frame translation events from the UL15 gene and that the 81-kDa full-length UL15 protein is required for DNA cleavage and packaging.

Animals↗
PubMed1991

[Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes].

The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C-negative regions, as well as between G-bands and R-bands, has been produced.

Animals↗
PubMed2005

Activation of the WUS gene induces ectopic initiation of floral meristems on mature stem surface in Arabidopsis thaliana.

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.

Arabidopsis↗
PubMed1983

An inverted repeat borders a fivefold amplification in satellite DNA.

One variant of a complex satellite DNA of the Bermuda land crab is significantly longer than the average repeat unit of the satellite. The extra DNA in the variant is accounted for by a fivefold tandem amplification of a 0.142-kilobase sequence. The amplified sequence is bounded by a tetranucleotide inverted repeat; the upstream arm of the inverted repeat is missing from two other variants of the satellite. The latter variants contain only one copy of a sequence that is closely related to the amplified sequence. By contrast, in several satellite DNA's of other organisms, extra DNA is inserted.

Animals↗
PubMed1983

Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids.

Marked changes in the uterine binding of oxytocin (OT) occur in rats at the time of parturition or after treatment of ovariectomized rats with estrogen or progesterone. To ascertain that these binding sites represent the biological receptors for OT, we measured the uterine response to OT in various groups of rats in which specific OT binding was also determined. Intact pregnant rats and rats ovariectomized on day 20 of gestation and treated thereafter with oil, estradiol benzoate (5 micrograms/24 h), progesterone (5 mg/24 h), or estradiol and progesterone together had indwelling balloons inserted on day 20 for the recording of uterine response to either iv bolus injections or iv infusions of OT. The uterus was removed 24-48 h after balloon insertion, and OT binding to the particulate fraction as well as nuclear estrogen and cytosolic estrogen receptor concentrations were determined. An inverse correlation (r2 = 0.758) was found between the concentration of OT-binding sites and the threshold dose of OT, and a linear correlation was found between the concentration of binding sites and the uterine activity induced by OT infusion (r2 = 0.852). We conclude, therefore, that the high affinity (Kd, 1-2 nM) binding sites for OT represent the physiological receptors. The concentration of these sites increased progressively during estrogen treatment. Progesterone completely inhibited this estrogen-induced rise. After ovariectomy, there was a modest, but significant, increase in OT receptor concentration which also was prevented by progesterone. The increase in OT receptor concentration was correlated with the estrogen receptor concentration in intact pregnant and estrogen-treated ovariectomized animals, but not in the other groups of animals. The apparent affinity of the receptors for OT was not significantly affected by hormone treatment. We conclude that the concentration of receptors is a major factor controlling the uterine responsiveness to OT, and that the receptor concentrations are regulated by ovarian hormones in a manner related to estrogen receptor activation. In addition, estrogen appeared to enhance the coupling of OT receptor occupancy to the tissue response to OT.

Animals↗
PubMed2001

Warning! Suctioning. A lung model evaluation of closed suctioning systems.

BACKGROUND: Closed system suctioning, CSS, has been advocated to avoid alveolar collapse. However, ventilator manufacturers indicate that extreme negative pressure levels can be obtained during closed system suctioning, impeding the performance of the ventilator. METHODS: Suctioning with a 12 or 14 Fr catheter with a vacuum level of -50 kPa was either performed with an open technology or a CSS, where the catheter is introduced through a tight-fitting connection through the endotracheal tube, EYT. The lung model was ventilated with a Servo 900C or 300 ventilator with an I:E ratio of 1:2, 1:1 and 2:1 and extrinsic positive end-expiratory pressure (PEEP) at 0 or 10 cm H20. Respiratory volumes and alveolar pressure were measured at the lung model alveolus. RESULTS: The initial suctioning flow was >40 l/min with a 14 Fr catheter. When inserting the catheter through a no. 7 ETT, PEEP rose from 11 to 23 cm H2O during volume control ventilation with an I:E ratio 1:1. During suctioning the alveolar pressure fell to 10 cm H2O below the set PEEP level. CSS during pressure control ventilation had fewer effects. Low tidal volumes, inverse I:E ratio and secretions in the tube resulted in pressures down to -92 cm H2O. CONCLUSION: CSS should not be used in volume control ventilation due to risk of high intrinsic PEEP levels at insertion of the catheter and extreme negative pressures during suctioning. Pressure control ventilation produces less intrinsic PEEP effect. The continuous positive airway pressure (CPAP) mode offers the least intrinsic PEEP during insertion of the catheter and least sub-atmospheric pressure during suctioning.

Air Pressure↗
PubMed2003

Molecular characterization of Brucella abortus chromosome II recombination.

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.

Animals↗
PubMed1990

Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E. coli: effects on in vivo function.

The selenocysteine-inserting tRNA (tRNA(Sec)) of E. coli differs in a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.

Anticodon↗
PubMed2006

Flexible needle steering for percutaneous therapies.

OBJECTIVE: A robotic system is presented for flexible needle steering and control in soft tissue. MATERIALS AND METHODS: Flexible needle insertion into a deformable tissue is modeled as a linear beam supported by virtual springs, where the stiffness coefficients of the springs can vary along the needle. Using this simplified model, the forward and inverse kinematics of the needle are solved analytically, thus enabling both path planning and path correction in real time. Given target and obstacle locations, the computer calculates the needle tip trajectory that will avoid the obstacle and hit the target. Using the inverse kinematics algorithm, the corresponding needle base maneuver needed to follow this trajectory is calculated. RESULTS: It is demonstrated that the needle tip path is not unique and can be optimized to minimize lateral pressure of the needle body on the tissue. Needle steering, i.e., the needle base movements that steer the needle tip, is not intuitive. Therefore, the needle insertion procedure is best performed by a robot. The model was verified experimentally on muscle and liver tissues by robotically assisted insertion of a flexible spinal needle. During insertion, the position and shape of the needle were recorded by X-ray. CONCLUSIONS: This study demonstrates the ability to curve a flexible needle by its base motion in order to achieve a planned tip trajectory.

Algorithms↗
PubMed1990

Ph-negative chronic myeloid leukemia: molecular analysis of ABL insertion into M-BCR on chromosome 22.

Leukemic cells from a patient with Ph-negative chronic myeloid leukemia (CML) had a normal karyotype. M-BCR was rearranged and chromosome in situ hybridization showed an ABL insertion between 5' and 3' M-BCR on an apparently normal chromosome 22. The association of 5' BCR and 3' ABL at the 5' junction of the chromosome 9 insert was typical of that found for the BCR-ABL fusion gene in other patients with the standard t(9;22) and CML. With an M-bcr-3' probe, we cloned and characterized a 3' junction fragment. Field inversion gel electrophoresis and chromosome in situ hybridization studies using a probe isolated from genomic DNA 5' of the junction showed that 3' M-BCR was joined to a region of chromosome 9q34 rich in repetitive sequences and lying some distance 3' of ABL. The chromosome 9 insert was at least 329 kilobases long and included 3' ABL and a larger portion of chromosome 9q34. Our results allowed us to exclude transposon- or retroviral-mediated insertion of ABL into chromosome 22. Instead, we favored a two-translocation model in which a second translocation reconstituted a standard t(9;22)(q34;q11) but left the chromosome 9 insert, including 3' ABL, in chromosome 22.

Chimera↗
PubMed1996

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.

Base Sequence↗
PubMed2004

Mechanistic analysis of hydroarylation catalysts.

Recently, two organometallic systems ([Ir(micro-acac-O)(acac-O,O)(acac-C(3))](2) and (Tp)Ru(CO)(Ph)(NCCH(3))) have been discovered that catalyze hydroarylation of unactivated olefins. Herein, we use density functional theory (B3LYP) to study the factors underlying this class of catalysts. In addition, we calculate the key steps for Rh, Pd, Os, and Pt with similar ligand sets. We previously showed there to be two key steps in the process: (i) insertion of a phenyl into the pi bond of a coordinating olefin, and (ii) C-H activation/hydrogen transfer of an unactivated benzene. An important discovery in these studies is that the barriers for these two steps are inversely correlated, complicating optimization of the overall process. However, herein we elucidate the causes of this inverse correlation, laying the foundation for the rational design of improved catalysts. Both steps are directly influenced by the accessibility of the higher 2-electron oxidation state, M(n) --> M(n+2). Systems with an easily accessible M(n+2) state activate C-H bonds easily but suffer from high energy insertions due to significant back-bonding. Conversely, systems without an easily accessible M(n+2) state have no debilitating back-bonding which makes insertion steps facile, but cannot effectively activate the C-H bond (leading instead to polymerization). The relationship between accessibility of the M(n+2) state and the amount of back-bonding in the coordinating olefin can be visualized by inspecting the hybridization of the coordinating olefin. Furthermore, we find a linear relation between this hybridization and the barrier to insertion. On the basis of these concepts, we suggest some modifications of the sigma framework expected to improve the rates beyond this linear correlation.

Journal Article↗