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PubMed2002

The role of biomechanical factors and HLA-B27 in magnetic resonance imaging-determined bone changes in plantar fascia enthesopathy.

OBJECTIVE: To study the role of biomechanical factors and HLA-B27 in plantar fasciitis. METHODS: T1-weighted and T2 spectral presaturation with inversion recovery (fat suppressed) magnetic resonance imaging (MRI) sequences of the plantar fascia insertion and adjacent bone were performed on 28 patients with plantar fasciitis; 17 had spondylarthropathy (SpA)-associated disease, and 11 had mechanically induced disease. The relationship between the degree of bone edema, scored on a semiquantitative scale (from absent to severe), and the patient's HLA-B27 status was determined. RESULTS: On MRI, edema within the soft tissue at the enthesis was evident in both groups. Bone edema in the adjacent calcaneum was evident in 64.7% (11 of 17) of patients with SpA and in 45% (5 of 11) of those with mechanically induced disease (P = 0.441). HLA-B27 was identified in 9 (53%) of the patients with SpA but in none (0%) of those with mechanically induced disease. All 6 of the SpA patients with extensive bone edema but none of the 5 SpA patients with mild bone edema were HLA-B27 positive (P = 0.002). CONCLUSION: The association of HLA-B27 with bone pathology in early enthesitis may have implications for a better understanding of the pathogenesis of SpA.

Achilles Tendon↗
PubMed2004

In silico comparative analysis reveals a mosaic conservation of genes within a novel colinear region in wheat chromosome 1AS and rice chromosome 5S.

Comparative RFLP mapping has revealed extensive conservation of marker order in different grass genomes. However, microcolinearity studies at the sequence level have shown rapid genome evolution and many exceptions to colinearity. Most of these studies have focused on a limited size of genomic fragment and the extent of microcolinearity over large distances or across entire genomes remains poorly characterized in grasses. Here, we have investigated the microcolinearity between the rice genome and a total of 1,500 kb from physical BAC contigs on wheat chromosome 1AS. Using ESTs mapped in wheat chromosome bins as an additional source of physical data, we have identified 27 conserved orthologous sequences between wheat chromosome 1AS and a region of 1,210 kb located on rice chromosome 5S. Our results extend the orthology described earlier between wheat chromosome group 1S and rice chromosome 5S. Microcolinearity was found to be frequently disrupted by rearrangements which must have occurred after the divergence of wheat and rice. At the Lr10 orthologous loci, microrearrangements were due to the insertion of mobile elements, but also originated from gene movement, amplification, deletion and inversion. These mechanisms of genome evolution are at the origin of the mosaic conservation observed between the orthologous regions. Finally, in silico mapping of wheat genes identified an intragenomic colinearity between fragments from rice chromosome 1L and 5S, suggesting an ancestral segmental duplication in rice.

Chromosome Aberrations↗
PubMed1990

Effects of culture and incubation conditions on membrane fluidity in monolayers of cultured cells measured as fluorescence anisotropy using trimethylammoniumdiphenylhexatriene (TMA-DPH).

Membrane fluidity of coverslip attached living cells was measured as fluorescence anisotropy using 5 microM trimethylammoniumdiphenylhexatriene (TMA-DPH) as fluorescent probe. Fluorescence anisotropy is inversely related to membrane fluidity. Cells were grown on glass coverslips that were inserted and directly incubated in quarz cuvettes. The coverslips were fixed with special holders at an angle of 30 degrees in respect to the incident light. Effects of incubation temperature, of cell growth and densities and of the ionic and nonionic composition of the incubation medium on membrane fluorescence anisotropy were measured. Membranes of growing cells were more fluid than those of stationary cells, while cell densities had no effect except at very low cell numbers. Calcium concentrations increasing from 0 to 8 mmol/l in the incubation medium proportionally decreased membrane fluidity. Hypotonicity of the incubation media increased membrane fluidity while hypertonicity compared to normotonicity had no effect. Differentiated human fibroblasts from different origins exhibited similar membrane fluidities. They were, however, different from those of rat cells. Membrane fluidity of rat brain tumor cells increased with age in culture while membrane fluidity of primary differentiating rat brain cells decreased in with age in culture. Measurement of fluorescence anisotropy in living cells attached to glass coverslips is a convenient tool to study effects of culture--as well as of environmental--conditions on membrane fluidity.

Animals↗
PubMed2005

Molecular evolution of the lysine decarboxylase-defective phenotype in Shigella sonnei.

Shigella, the etiological agent of the bacillary dysentery, belongs to the extremely diverse species of Escherichia coli. In the evolutionary route of Shigella from commensal E. coli ancestors towards a pathogenic lifestyle, the critical events have been the acquisition of the pINV plasmid, through horizontal transfer and the inactivation of pre-existing genes. These so-called pathoadaptive mutations affect the expression of genes negatively interfering with the newly acquired functions necessary for the colonization of the host niche. Cadaverine, a small polyamine resulting from decarboxylation of lysine, has been shown to hamper the full expression of Shigella invasiveness mainly by altering the inflammatory response. Recent analysis of the evolution of the Shigella and enteroinvasive E. coli (EIEC) cad region indicates that silencing of the cad locus has been attained with several strategies. The increasing relevance of S. sonnei in both, developing and industrial countries, prompted us to analyze the molecular origin of the LDC- phenotype in these strains. The results obtained on several S. sonnei strains reveal that despite the difference in geographic origin and antibiotic resistance patterns, all the strains have undergone the same modifications. Multiple IS insertions into the cadBA operon have interrupted gene continuity without inducing deletions or inversions of the cadA and cadB genes which are remained entirely conserved. Moreover, by functional analysis we show that all the strains carry a defective cadC gene, thus strengthening the hypothesis that inactivation of the regulatory cadC gene might have been the first step towards a complete lack of the cad locus.

Bacterial Proteins↗
PubMed1999

Meiotic studies in Dysdercus Guérin Meneville 1831 (Heteroptera: Pyrrhocoridae). I. Neo-XY in Dysdercus albofasciatus Berg 1878, a new sex chromosome determining system in Heteroptera.

The genus Dysdercus Guérin Méneville 1831 represents the only taxon within the family Pyrrhocoridae in the New World. Based on morphological features, it has been suggested that American species derived from immigrants from the Old World, most probably from the Ethiopian Region. So far, 10 species from Dysdercus, including six species from the Old World and four species from the Neotropical Region have been cytogenetically analyzed. As is characteristic of Heteroptera, they possess holokinetic chromosomes and a prereductional type of meiosis. While the X1X20 sex chromosome system has been reported in all cytologically analyzed species of Dysdercus from the Old World, the system X0 has been found in all but one species from the New World, regardless of the number of autosomes in the complement. In the present study the male meiosis of D. albofasciatus Berg 1878 was studied in specimens from four different populations from Argentina. The diploid chromosome number was found to be 2n = 10 + neo-XY. The neo-X shows at each subterminal region a positively heteropycnotic and DAPI-bright segment which corresponds to the ancestral X-chromosome. The origin of this neo-XY system involved, most probably, a subterminal insertion of the ancestral X chromosome in an autosome, followed by a large inversion, which included part of the original X chromosome.

Animals↗
PubMed1990

Disruption of formin-encoding transcripts in two mutant limb deformity alleles.

The recent identification of a gene residing at the mouse limb deformity (ld) locus permits us to test the hypothesis that disruption of this gene is responsible for an inherited anomaly affecting embryonic pattern formation. The gene gives rise to alternatively processed messenger RNAs that can be translated as a family of related protein products, termed the formins. We have now analysed transcripts from this gene in four independently isolated mutant alleles. In two of these, the ldHd allele (created by insertion of a transgene) and the ldIn2 allele (created by a translocation-inversion involving mouse chromosomes 2 and 17), a common subset of ld transcripts is abolished, but others are apparently unaltered. The correlation of altered transcripts in two independent ld mutants strongly supports the notion that one or more altered formins is responsible for the observed phenotype. That the defect is limited to the limb and kidney, despite expression of ld mRNA in other unaffected organs, suggests that these mutant alleles represent only partial loss of ld function.

Alleles↗
PubMed1991

Positive transcriptional regulation of an iron-regulated virulence gene in Vibrio cholerae.

We have previously described a virulence gene in Vibrio cholerae (irgA) that is more than 850-fold regulated in response to iron. Negative regulation of irgA by iron occurred at the transcriptional level, and there was a dyad symmetric nucleotide sequence in the vicinity of the irgA promoter homologous to the Fur binding site in Escherichia coli. When irgA was cloned into E. coli, we showed that transcription of irgA required 900 base pairs of DNA upstream of the irgA promoter that contained an open reading frame in inverse orientation to irgA. In the present study, we show that this upstream region of DNA encodes a gene in inverse orientation to irgA (named irgB) that is also negatively regulated by iron. Insertional inactivation of irgB on the V. cholerae chromosome leads to loss of expression of a chromosomal irgA'-'phoA fusion (in which the primes indicate truncated genes), which is restored to normal by provision of irgB on a plasmid in trans. DNA sequencing of irgB shows that the protein product (IrgB) is homologous to the LysR family of positive transcriptional activators, and secondary structure analysis of IrgB predicts a helix-turn-helix DNA binding motif. The promoters of irgB and irgA are divergent but overlap each other and the previously defined Fur-binding site. We propose a model for iron regulation of irgA expression in V. cholerae. In the presence of sufficient iron, transcription of both irgA and irgB is negatively regulated by a Fur-like protein. In low iron conditions, negative regulation of transcription is removed, and production of IrgB leads to positive transcriptional activation of irgA. It seems likely that the high induction ratio of irgA expression under low- and high-iron conditions (850-fold) relates to the fact that its cognate positive transcriptional activator (irgB) is itself negatively regulated by iron.

Alkaline Phosphatase↗
PubMed1990

Quantitative effects of P elements on hybrid dysgenesis in Drosophila melanogaster.

Genetic analyses involving chromosomes from seven inbred lines derived from a single M' strain were used to study the quantitative relationships between the incidence and severity of P-M hybrid dysgenesis and the number of genomic P elements. In four separate analyses, the mutability of snw, a P element-insertion mutation of the X-linked singed locus, was found to be inversely related to the number of autosomal P elements. Since snw mutability is caused by the action of the P transposase, this finding supports the hypothesis that genomic P elements titrate the transposase present within a cell. Other analyses demonstrated that autosomal transmission ratios were distorted by P element action. In these analyses, the amount of distortion against an autosome increased more or less linearly with the number of P elements carried by the autosome. Additional analyses showed that the magnitude of this distortion was reduced when a second P element-containing autosome was present in the genome. This reduction could adequately be explained by transposase titration; there was no evidence that it was due to repressor molecules binding to P elements and inhibiting their movement. The influence of genomic P elements on the incidence of gonadal dysgenesis was also investigated. Although no simple relationship between the number of P elements and the incidence of the trait could be discerned, it was clear that even a small number of elements could increase the incidence markedly. The failure to find a quantitative relationship between P element number and the incidence of gonadal dysgenesis probably reflects the complex etiology of this trait.

Animals↗
PubMed1991

Allelic interactions at the nivea locus of Antirrhinum.

Most null alleles at the nivea (niv) locus are recessive to Niv+ and, when homozygous, give white flowers rather than the red of the wild type. In contrast, the niv-571 allele is semidominant; although it gives white flowers when homozygous, very pale flowers result when this allele is heterozygous with NIV+. We showed that in heterozygotes, niv-571 acts in trans to inhibit expression of its Niv+ homology 25-fold to 50-fold. The inhibition is reversible after meiosis and partially reversible somatically. The niv-571 allele carries a transposable element Tam3 insertion and three truncated copies of the niv gene, one copy being in inverse orientation. Analysis of two further niv alleles, niv-572 and niv-527, showed that excision of Tam3 from niv-571 does not affect the ability of the allele to repress Niv+ and that one truncated niv copy alone is insufficient to confer semidominance. The detailed structures of various semidominant niv alleles suggest that their effects in trans are not readily explained by production of antisense RNA but are more easily reconciled with a direct recognition/interaction between homologous genes, reminiscent of cosuppression and transvection phenomena described in other systems.

Acyltransferases↗
PubMed2000

impA, a gene coding for an inner membrane protein, influences colonial morphology of Actinobacillus actinomycetemcomitans.

Directed mutagenesis of a gene coding for a membrane protein of the periodontopathogen Actinobacillus actinomycetemcomitans was achieved by conjugation. The gene was disrupted by insertion of an antibiotic cassette into a unique endonuclease restriction sequence engineered by inverse PCR. The disrupted gene was cloned into a conjugative plasmid and transferred from Escherichia coli to A. actinomycetemcomitans. The allelic replacement mutation resulted in the loss of a 22-kDa inner membrane protein. The loss of this protein (ImpA) resulted in changes in the outer membrane protein composition of the bacterium. Concurrent with the mutation in impA was a change in the pattern of growth of the mutant bacteria in broth cultures. The progenitor bacteria grew as a homogeneous suspension of cells compared to a granular, autoaggregating adherent cell population described for the mutant bacteria. These data suggest that ImpA may play a regulatory role or be directly involved in protein(s) that are exported and associated with colony variations in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1990

Disruption of a rhodaneselike gene results in cysteine auxotrophy in Saccharopolyspora erythraea.

A 3,373-base-pair DNA segment from a clone fortuitously isolated from Saccharopolyspora erythraea by hybridization to an oligodeoxynucleotide probe was sequenced. Computer-assisted analysis of the nucleotide sequence reveals three closely linked Streptomyces open reading frames plus a fourth converging on the others. The deduced product of one of them, ORF2, shows considerable similarity to bovine liver rhodanese. orf2, and the closely linked orf3 located just downstream of it, were disrupted by insertion of an apramycin resistance cassette into the orf2 coding sequence along with inversion of the fragment carrying most of orf2 and orf3 via two successive recombinational events in the wild-type strain. The mutant strain thus created contains wild-type levels of rhodanese activity but cannot grow on minimal medium. It is a cysteine auxotroph, capable of utilizing efficiently only thiosulfate among the inorganic sulfur sources tested. orf2 has been designated cysA. The possible role of the rhodaneselike cysA gene product in thiosulfate formation is discussed.

Base Sequence↗
PubMed2005

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.

Amino Acid Sequence↗
PubMed1995

The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences.

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.

Animals↗
PubMed2000

Campylobacter fetus sap inversion occurs in the absence of RecA function.

Phase variation of Campylobacter fetus surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique sap promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the C. fetus sap inversion system is RecA dependent. When we challenged a pregnant ewe with a recA mutant of wild-type C. fetus (strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the aphA insertion into recA was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the sap locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these recA mutants the sap inversion frequency is reduced by 2 to 3 log(10) units compared to that in the wild type. Thus, although the major sap inversion pathway in C. fetus is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.

Animals↗
PubMed2005

[Regulatory effect of STAT5 decoy oligonucleotides on trans-activation of bcl-x gene promoter in K562 cells].

BACKGROUND & OBJECTIVE: Constitutive activation of signal transducers and activators of transcription 5 (STAT5) plays an important role in malignant transformation of chronic myeloid leukemia (CML) cells. This study was to explore regulatory effect of STAT5 decoy oligonucleotides (ODNs) on trans-activation of its downstream target bcl-x gene in K562 cells. METHODS: STAT5 decoy ODNs, mismatched ODNs (M-ODNs), and FAM-decoy ODNs were designed and synthesized. FAM-decoy ODNs were used as control, and transfected into K562 cells by cationic liposomes, analyzed by flow cytometry (FCM) and fluorescent inversive microscopy. The bcl-x promoter fragment acquired by polymerase chain reaction (PCR) was inserted into pGL3-basic to construct luciferase report plasmid pGL3b-bclxp, which was co-transfected with decoy ODNs or M-ODNs into K562 cells. The activity of luciferase was detected. After transfection of decoy ODNs, and M-ODNs, expression of bcl-xL mRNA in K562 cells was detected by reverse transcription-PCR (RT-PCR),cell apoptosis was detected by FCM. RESULTS: FAM-decoy ODNs were incorporated into K562 cells in a dose-dependent manner. The incorporation efficiency reached 99.1% at the concentration of 4 micromol/L 24 h after transfection, and green fluorescence could be observed in cells under fluorescent inversive microscope. The luciferase activity was significantly lower in STAT5 decoy ODNs group than in control group [(181.48+/-204.46) RLU/microg protein vs. (675.26+/-62.91) RLU/mug protein, P < 0.05], but that of M-ODNs group [(632.07+/-98.95) RLU/microg protein] has no significant difference with that of control group (P > 0.05). mRNA level of bcl-xL was decreased by STAT5 decoy ODNs, but not by M-ODNs. Moreover, Sub G1 peak was detected in STAT5 decoy ODNs group by FCM. CONCLUSION: Transfection of STAT5 decoy ODNs can down-regulate the trans-activation of bcl-x in K562 cells.

Apoptosis↗
PubMed1999

MR-guided RF thermal ablation of the kidney in a porcine model.

OBJECTIVE: The purpose of the study was to evaluate the ability of MR imaging to reveal RF interstitial thermotherapy in the porcine kidney, as a model for future human trials, and to provide guidance for RF probe insertion. SUBJECTS AND METHODS: Ten MR-guided RF ablations were performed in the kidneys of three pigs. A 17-gauge monopolar RF probe electrode was inserted into the renal cortex using MR guidance, and RF was applied for 10 min. After postprocedure imaging (T2-weighted, turbo short inversion time inversion recovery [STIR], and T1-weighted sequences), the kidneys were harvested for pathologic examination. RESULTS: Successful RF probe placement was accomplished in all cases; the interventional procedure time for probe insertion ranged from 4 to 15 min. The thermal lesion size varied from 7 to 14 mm perpendicular to the probe track and was best seen on turbo STIR images. Turbo STIR had the highest renal cortex-to-RF thermal lesion contrast-to-noise ratio with an average of 4.4 +/- 3.5. Turbo STIR imaging never overestimated pathologic lesion diameter with a mean difference of 1.5 +/- 1.4 mm. Three subcapsular hematomas occurred. which could be detected immediately on postprocedure imaging. CONCLUSION: MR-guided RF thermal ablation in the porcine kidney was found to be feasible, but minor complications occurred. Induced thermal lesion size was best monitored using turbo STIR images. In the future, RF ablation may offer an alternative treatment option for renal cancer.

Animals↗
PubMed1998

Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn49 system of Drosophila melanogaster.

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement.

Animals↗