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Mechanisms of spiroplasma genome variation associated with SpV1-like viral DNA inferred from sequence comparisons.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral sequences in genome evolution, we examined available nucleotide sequences of viruslike elements in the S. citri chromosome. Comparison of integrated and nonintegrated sequences of spiroplasma virus SpV1-C74 DNA suggested that it is an encapsidated form of the circular transposition intermediate belonging to an insertion sequence (IS3) family member. One SpV1-C74 viral DNA fragment was identified as interrupting the remains of a DNA adenine modification methylase gene. A viral DNA insertion of SpV1-R8A2 B DNA had hallmarks of having suffered an internal deletion by a site-specific recombination system. Homologous recombination likely was responsible for several deletions within viral DNA. A homologous recombination event was inferred between part of a viral DNA insertion and a similar chromosomal sequence. Dispersed sequences from SpV1-like C4 open reading frames (ORFs) were identified as involved in a complex deletion-inversion event. Thus, SpV1-like sequences likely have altered spiroplasma genomes by inserting within active genes, destroying their function, by providing targets for site-specific recombination, by mediating deletions of sequences adjacent to their integration sites, and by providing targets for homologous recombination, leading to inversions.

Amino Acid Sequence↗

Nucleotide sequence comparison of a chromosome rearrangement on human chromosome 12 and the corresponding ape chromosomes.

Chromosome rearrangement has been considered to be important in the evolutionary process. Here, we demonstrate the evolutionary relationship of the rearranged human chromosome 12 and the corresponding chromosome XII in apes (chimpanzee, bonobo, gorilla, orangutan, and gibbon) by examining PCR products derived from the breakpoints of inversions and by conducting shotgun sequencing of a gorilla fosmid clone containing the breakpoint and a "duplicated segment" (duplicon). We confirmed that a pair of 23-kb duplicons flank the breakpoints of inversions on the long and short arms of chimpanzee chromosome XII. Although only the 23-kb duplicon on the long arm of chimpanzee chromosome XII and its telomeric flanking sequence are found to be conserved among the hominoids (human, great apes, and gibbons), the duplicon on the short arm of chimpanzee chromosome XII is suggested to be the result of a duplication from that on the long arm. Furthermore, the shotgun sequencing of a gorilla fosmid indicated that the breakpoint on the long arm of the gorilla is located at a different position 1.9 kb from that of chimpanzee. The region is flanked by a sequence homologous to that of human chromosome 6q22. Our findings and sequence analysis suggest a close relationship between segmental duplication and chromosome rearrangement (or breakpoint of inversion) in Hominoidea. The role of the chromosome rearrangement in speciation is also discussed based on our new results.

Animals↗

Memory in bacteria and phage.

Whenever the state of a biological system is not determined solely by present conditions but depends on its past history, we can say that the system has memory. Bacteria and bacteriophage use a variety of memory mechanisms, some of which seem to convey adaptive value. A genetic type of heritable memory is the programmed inversion of specific DNA sequences, which causes switching between alternative patterns of gene expression. Heritable memory can also be based on epigenetic circuits, in which a system with two possible steady states is locked in one or the other state by a positive feedback loop. Epigenetic states have been observed in a variety of cellular processes, and are maintained by diverse mechanisms. Some of these involve alternative DNA methylation patterns that are stably transmitted to daughter molecules and can affect DNA-protein interactions (e.g., gene transcription). Other mechanisms exploit autocatalytic loops whereby proteins establish the proper conditions for their continued synthesis. Template polymers other than nucleic acids (e.g., components of the cell wall) may also propagate epigenetic states. Non-heritable memory is exemplified by parasitic organisms that bear a signature of their previous host, such as host-controlled modification of phage DNA or porin hitchhiking in predatory bacteria. The heterogeneous nature of the examples known may be indicative of widespread occurrence of memory mechanisms in bacteria and phage. However, the actual extent, variety and potential selective value of prokaryotic memory devices remain open questions, still to be addressed experimentally.

Bacteria↗

An apparent unidirectional influx constant for manganese as a measure of myocardial calcium channel activity.

PURPOSE: To develop an in vivo MR method for evaluation of myocardial calcium channel activity through quantification of apparent unidirectional manganese influx constants following manganese dipyridoxyl-diphosphate (MnDPDP) infusions. MATERIALS AND METHODS: A total of 10 healthy volunteers were divided in two groups, and received 5 micromol of MnDPDP per kg of body weight intravenously in a 1.5 Tesla scanner over five or 30 minutes, respectively. A fast inversion recovery gradient echo sequence was used to estimate pre- and postcontrast R1 values and to measure signal changes following infusions. By assuming equal longitudinal relaxivity (r1) of the contrast in all tissue compartments, signal changes in blood and myocardial tissue yielded temporal input and tissue contrast concentrations respectively. Through a two-tissue compartment model, apparent unidirectional influx constants (Ki) for myocardial manganese accumulation were estimated. RESULTS: Consistent values for Ki in left ventricular wall were found, with a mean value of 5.96 mL/100 g/minute (SD=0.49; N=10). No statistical significant differences in Ki were found between the two infusion groups. CONCLUSION: Since unidirectional manganese accumulation depends upon intact myocyte membranes with functioning calcium channels, the use of unidirectional manganese influx rates may be a valuable research tool for in vivo studies of myocyte functioning in myocardial disease.

Adult↗

Test-retest reproducibility of quantitative CBF measurements using FAIR perfusion MRI and acetazolamide challenge.

The reproducibility of quantitative cerebral blood flow (CBF) measurements using MRI with arterial spin labeling and acetazolamide challenge was assessed in 12 normal subjects, each undergoing the identical experimental procedure on two separate days. CBF was measured on a 1.5T scanner using a flow-sensitive alternating inversion recovery (FAIR) pulse sequence, performed both at baseline and 12 min after intravenous administration of acetazolamide. T(1) was measured in conjunction with the FAIR scan in order to calculate quantitative CBF. The CBF maps were segmented to separate gray matter (GM) from white matter (WM) for region-of-interest (ROI) analyses. Post- acetazolamide CBF values (ml/100 g/min, mean +/- SD) of 87.5 +/- 12.5 (GM) and 46.1 +/- 10.8 (WM) represented percent increases of 37.7% +/- 24.4% (GM) and 40.1% +/- 24.4% (WM). Day-to-day differences in baseline CBF were -1.7 +/- 6.9 (GM) and -1.4 +/- 4.7 (WM) or, relative to the mean CBF over both days for each subject, -2.5% +/- 11.7% (GM) and -3.8% +/- 13.6% (WM) Day- to-day differences in absolute post-ACZ CBF increase were -2.5 +/- 6.8 (GM) and 2.7 +/- 9.4 (WM) or, relative to the mean CBF increase over both days for each subject, -4.7% +/- 13.3% (GM) and 9.1% +/- 26.2% (WM). Thus, FAIR- based CBF measurements show satisfactory reproducibility from day to day, but with sufficient variation to warrant caution in interpreting longitudinal data. The hemispheric asymmetry of baseline CBF and post-acetazolamide CBF increases varied within a narrower range and should be sensitive to small changes related to disease or treatment.

Acetazolamide↗

Paramagnetic contrast enhancement at 0.02 T: an experimental study using Gd-DOTA in normal and hydronephrotic kidneys.

Both normal and experimentally hydronephrotic rabbits were imaged at 0.02 T using partial saturation (PS 160/30) and inversion recovery (IR 1000/200/40) sequences. The signal intensity of normal renal medulla and cortex markedly increased after the injection of 0.1 mmol/kg of Gd-DOTA. In the unilateral total hydronephrosis the dilated renal pelvis did not contrast enhance after 15 and 35 min of Gd-DOTA injection. The enhancement pattern was similar in 1- and 3-week-old hydronephrosis. The effect of Gd-DOTA on renal T1 times at 0.02 T was studied using rats. Fifteen minutes after the Gd-DOTA injection (0.1 mmol/kg) the T1 times of excised rat kidneys decreased from 311 to 90 ms. The authors conclude that the enhancement of the MR signal of the kidney by Gd-DOTA at an ultralow magnetic field (0.02 T) is similar to its enhancement at higher fields (greater than 0.15 T).

Animals↗

Improvement of spectral resolution in shift-reagent-aided 23Na NMR spectroscopy in the isolated perfused rat heart system.

The level of intracellular sodium (Nai) is maintained at approximately 14 mM in healthy myocytes. When myocytes are damaged, Nai increases and therefore the level of Nai may be a means of evaluating myocardial cell integrity. A particularly useful method to monitor Nai levels is 23Na NMR spectroscopy. However, because of the isochronous nature of the extracellular sodium (Nao) and Nai NMR signals, paramagnetic lanthanide shift reagents (LSR), such as dysprosium triphosphate, Dy(PPP)7-(2), have been used to shift the Nao signal. This reveals the unshifted Nai signal and allows the NMR monitoring of Nai in isolated perfused hearts and other systems. A major shortcoming of this method (the "shift-only" method) is in the need to minimize the Nao signal by not submerging the perfused hearts in Na(+)-containing buffer. An equally undesirable alternative is the utilization of relatively high concentrations of LSR to shift a large Nao signal sufficiently to enable reasonable resolution and quantitation of Nai. We present here a method, the "shift-relaxation" method, which is a combination of using a mixture of Dy(PPP)7-(2), a shift reagent, and gadolinium triphosphate, Gd(PPP)7-(2), a relaxation agent, with data acquisition using an inversion-recovery (IR) pulse sequence. This combination allows differentiation between Nao and Nai by the difference in their respective T1 values in addition to the shift between them. With this technique we can selectively minimize the extracellular signal and therefore minimize the need for a large Dy-induced shift, as well as allow data acquisition on a heart submerged in Na(+)-containing perfusate. The resulting improved discrimination between Nai and Nao at relatively low levels of LSR should be helpful for ultimate in vivo applications and potential clinical applications, where a lower dose of LSR also means a decreased possibility of physiologically deleterious effects. Also included in this paper is a method for the quick determination of an accurate 180 degrees pulse which is required for the optimization of the IR method.

Animals↗

Continuous measurement of cell volume changes in perfused rat salivary glands by proton NMR.

Changes in intracellular and extracellular water content have been measured in perfused rat salivary glands by repetitive application of an inversion recovery (IR) pulse sequence. The relaxation reagent Gd-DTPA (10 mM) was included in the perfusate so that the intracellular and extracellular water proton signals could be distinguished by their different longitudinal relaxation times. Changes in water content in response to altered perfusion pressure and perfusate osmolarity were determined at 30-s intervals and indicated a clear separation of the intracellular and extracellular components. Using a modification of the IR pulse sequence, changes in intracellular water content were also measured at 6-s intervals. With this time resolution, differences in the rates of cell shrinkage in response to hyperosmotic perfusates and the secretomotor agonist acetylcholine were observed. The results suggest that this approach offers a relatively noninvasive method for studying cell volume regulation in intact, perfused tissues and organs.

Acetylcholine↗

Pulmonary perfusion: qualitative assessment with dynamic contrast-enhanced MRI using ultra-short TE and inversion recovery turbo FLASH.

The accurate assessment of pulmonary perfusion is especially important in the evaluation of patients with suspected pulmonary embolism, a common and potentially lethal disorder that can be treated by aggressive anticoagulation. In this study, we demonstrate for the first time the use of MR to image pulmonary perfusion in humans by using dynamic imaging after contrast administration. The technique, which uses an inversion recovery turbo FLASH sequence with ultrashort TE (1.4 ms) and 1-s temporal resolution, was tested in a series of eight healthy subjects and in a porcine model of pulmonary embolism. After the administration of gadopentetate dimeglumine in humans and animal models, there was serial enhancement of the systemic veins, right atrium, right ventricle, and pulmonary arteries. The pulmonary arterial tree was visualized beyond the segmental branches, followed by a gradual diffuse increase in signal intensity of the lung parenchyma over a period of 4.0-7.0 s. Pulmonary circulation times ranged from 3.0-3.4 s. Whereas a high dose (20 or 40 ml) of contrast agent tended to produce the most intense parenchymal enhancement, a low dose (5 ml) was best for showing recirculation. In the animal model, a perfusion defect due to a pulmonary embolus was clearly shown and confirmed by cine angiography. It is concluded that MRI of lung perfusion is feasible. With further development, perfusion MRI could eventually have a significant clinical role in the diagnostic evaluation of pulmonary embolism.

Animals↗

Estimating NAA in cortical gray matter with applications for measuring changes due to aging.

N-acetylaspartic acid (NAA), a prominent peak in the proton spectrum, is an amino acid thought to be present almost exclusively in neurons and their dendritic and axonal extensions. While 1H MRS studies are showing promise in identifying NAA deficits in different patient groups, unwanted lipid signal from subcutaneous fat surrounding the skull, and necessarily large voxels have limited investigators' ability to assess NAA in cortical gray matter. Here we report a technique developed to derive estimates of NAA signal from cortical gray matter. This approach uses an inversion recovery imaging pulse sequence with a long TE to suppress lipid signal from the scalp and information from concurrently obtained structural MR images to determine the CSF, white and gray matter composition of each spectroscopic voxel. A regression analysis is then used to estimate what NAA levels would be in "pure" white and gray matter voxels. This technique has been applied to demonstrate reduced NAA gray/white levels in the brains of five healthy older compared with five healthy younger women.

Adult↗

Distribution of the fungal transposon Restless: full-length and truncated copies in closely related strains.

The fungal transposon Restless of Tolypocladium inflatum ATCC 34921 is a member of the hAT family of mobile DNA elements. In order to study the distribution of this transposon we have looked at 13 fungal strains, most of which are taxonomically related to strain ATCC 34921. Three strains, which show identical banding patterns in a comparative RAPD analysis with strain ATCC 34921, similarly carry multiple copies of Restless. In addition, 1 T. inflatum strain and 2 Beauveria nivea strains contain only a few or even single copies of the transposon. Inverse PCR and DNA sequencing analysis revealed that 1 strain contains a nonmobile truncated version of the element, while the other one harbors a full-length transposon copy which was named Restless-2. The presence of a single transposon copy of Restless in a defined Beauveria strain indicates recent acquisition of this transposon, since class II transposons usually occur in several copies per haploid genome. Notably, the corresponding genomic location is not occupied by a transposon copy in strain ATCC 34921.

Base Sequence↗

A new two-dimensional pulse sequence for T(2)* measurements of protons in (13)C isotopomers.

A new two-dimensional pulse sequence for T(2)* measurement of protons directly coupled to (13)C spins is proposed. The sequence measures the tranverse relaxation time of heteronuclear proton single-quantum coherence under conditions of free precession and is therefore well suited to evaluate relaxation losses of proton magnetization during preparation delays of heteronuclear pulse experiments in analytical NMR. The relevant part of the pulse sequence can be inserted as a "building block" into any direct or inverse detecting H,C correlation pulse sequence if proton spin-spin relaxation is to be investigated. In this contribution, the building block is inserted into a HETCOR as well as into a HMQC pulse sequence. Experimental results for the HETCOR-based sequence are given.

Journal Article↗

Isolation and characterization of chromosomal mTn 10 insertion mutations affecting K88 fimbriae production in Escherichia coli.

mTn 10 transposon mutagenesis of Escherichia coli producing K88 fimbria was carried out in order to identify host factors involved in the regulation of the fae (K88) operon and the production of K88 fimbriae. Five independent chromosomal insertion mutants were obtained which showed an increased expression of K88 fimbriae. Inverse PCR and nucleotide sequencing were carried out to characterize the mutations. One insertion affected the Ipp gene, encoding the major outer membrane lipoprotein. Another mutation was found to be located in the Irp gene, encoding the 'global' regulatory protein Lrp (leucine responsive regulatory protein). A third mutant was found to affect the expression of rfaF, encoding heptosyltransferase II, which resulted in a partially wild-type and partially Re-Rd1 type of LPS. A fourth mutation affected sseB, a gene involved in serine-sensitivity of E. coli cells. Another mutant contained an insertion in an unknown region of the E. coli genome. The mutants were further characterized with respect to K88 as well as K99 fimbriae production.

Amino Acid Sequence↗

Functional properties of the anaerobic responsive element of the maize Adh1 gene.

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promoter. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position -90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/delta 35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.

Alcohol Dehydrogenase↗

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another Km transposon, Tn903.

A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.

Base Sequence↗

Mapping of normal brain maturation in infants on phase-sensitive inversion--recovery MR images.

It is illustrated that phase-sensitive inversion-recovery MR images are particularly well suited for the monitoring of brain maturation and myelination in the neonate and young infant. Provided appropriate timings are applied with the inversion-recovery MR pulse sequence, the myelinated areas show up as bright spots in the phase-sensitive images. The chronology of the appearance, and the location of these hyperintense zones correlate well with the chronology of brain maturation, as assessed by other means. In particular, the progressive functional capabilities of the infant correlate well with the progress of myelination, as exhibited by the MR images.

Brain↗

Modifications in the B10 and B26-30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor.

Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40-60% relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5-2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28-29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31-32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin.

Amino Acid Sequence↗

Improved detection of cortical and subcortical tubers in tuberous sclerosis by fluid-attenuated inversion recovery MRI.

We carried out fluid-attenuated inversion recovery (FLAIR) pulse sequences with long repetition and echo times in seven children with tuberous sclerosis, and compared them with conventional spin-echo (SE) sequences. FLAIR images exhibited higher sensitivity than conventional SE images to cortical and subcortical tubers. The low signal intensity of cerebrospinal fluid on FLAIR images allowed more accurate delineation of the cortical and subcortical tubers. However, T1-weighted imaging was still superior for delineation of subependymal nodules.

Adolescent↗