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PubMed1990

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.

Amino Acid Sequence↗

PubMed1995

Improvement of right hemispheric functions in a child with Gilles de la Tourette's syndrome by weak electromagnetic fields.

Gilles de la Tourette's syndrome (GTS) is a chronic, familial neuropsychiatric disorder of unknown etiology characterized clinically by the occurrence of motor and vocal tics and by the presence of a variety of neurobehavioral and neurocognitive abnormalities including hyperactivity, self-multilatory behavior, obsessive-compulsive behavior, learning disabilities, and conduct disorder. On the basis of neuropsychological assessments it has been suggested that GTS is associated with greater right than left hemispheric dysfunction which accounts for decrements in visuospatial, visuoconstructional and visuomotor skills in these patients. Recent case studies have demonstrated that extracranial application of electromagnetic fields (EMFs) in the picotesla (pT) range intensity improves visuospatial and visuoperceptive functions in patients with neurodegenerative disorders including Parkinson's disease, multiple sclerosis and Alzheimer's disease. I now present a 6 1/2 year old boy with GTS in whom this treatment modality produced, in addition to symptomatic behavioral improvement, also improvement in visuoconstructional and visuomotor skills as evidenced on various drawing tasks particularly copy of the Rey-Osterrieth Complex Figure, a task which is especially vulnerable to right hemispheric functions. These findings suggest that pT range EMFs may be useful for the treatment of GTS and related disorders and also reverse some of the cognitive impairments associated with the disease which are related to right hemispheric dysfunction and which contribute to learning disabilities in these patients.

Brain↗

PubMed1998

Polymerase chain reaction amplification of long DNA targets: application to analysis of chromosomal translocations in human B-cell tumors (review).

Chromosomal translocations in human leukemias generate fusion transcripts containing messages from two genes involved in the translocation, and these have been the targets for reverse transcriptase-mediated polymerase chain reaction (PCR). In contrast, many of the translocations in B-cell tumors involve immunoglobulin gene (IG) loci, and coding regions of the oncogenes on partner chromosomes are not interrupted by the translocation. Therefore, targets for PCR amplification are single-copy oncogene/IG fusion sequences within the complex genomic DNA. We present here a novel strategy for detection of translocations in B-cell tumors on the basis of long-distance (LD-) PCR that is capable of amplifying up to 30 kb of DNA. LD-PCR is a general method using primer pairs designed for distinctive regions of IG and oncogenes involved in translocations, and amplifying long DNA fragments encompassing the oncogene/IG junction. LD-PCR is capable of detecting virtually all the important translocations in B-cell tumors, including t(8;14)(q24;q32), t(14;18)(q32;q21), t(3;14)(q27;q32) and its variants. We show here that LD-PCR can substitute for time-consuming Southern blot hybridization in the rapid detection of these translocations. Furthermore, as amplified fragments obtained by LD-PCR contained exons and flanking sequences of the oncogenes and IGs, restriction analysis and nucleotide sequencing of the products refined the characteristics of translocations.

Chromosome Mapping↗

PubMed1998

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.

Bromovirus↗

PubMed2005

Folded RNA from an intron of one gene might inhibit expression of a counteracting gene.

Homeostatic maintenance of mRNA levels including prompt availability of mRNAs for translation in response to changing protein demands might be partly enabled by a system of combinatorial controls involving noncoding RNA blocking agents. This article proposes a specific version of that control mechanism, namely, a double-stranded RNA folding from transcription of an intron of one gene might and leading to an agent that inhibits mRNA of a counteracting gene. Thus transcription of the first gene would automatically repress translation of the second. On the basis of a bioinformatics search, we suggest a possible example, namely, that pro-apoptosis gene PAR4 might inhibit anti-apoptosis gene XIAP. Part of an intron from PAR4 folds to form a large, stable hairpin, and reverse complement of the hairpin stem (with approximately 280 nucleotides) matches a sub-sequence of an exon in XIAP. This would be part of an efficient system to drive initiated apoptosis to its conclusion. Figuratively speaking, it replaces two control knobs with one. Since repeats, some with many thousands of copies, occur throughout the genome with complex distributions, care must be taken before asserting that the presence of any repeat in any gene is significant. Our apoptosis example involves repeats, so experimental verification is needed and planned. However, if it is found that the noncoding RNA by-product of one gene folds into a hairpin that is processed into an agent that inhibits a counteracting gene, then the same type of control unit might be found extensively among counteracting families of genes of many types.

Animals↗

PubMedDate not supplied

[Frontal dysfunction in idiopathic Parkinson's disease].

INTRODUCTION: In his description of the disease in his original work, James Parkinson claimed that the 'senses remained intact', but later reports began to identify cognitive impairment that ranged from dementia to barely identifiable subclinical deteriorations. Research carried out in recent decades has revealed that cognitive disorders form part of the clinical symptoms of Parkinson's disease (PD) and point to the frontal lobes as being the most affected areas; a great deal of controversy, however, still surrounds their definition, epidemiology and pathology. AIM: To determine and classify the frontal deficits associated to this disease and to relate this cognitive performance with certain characteristics of the disease. SUBJECTS AND METHODS: The sample utilised in the study was made up of 222 subjects divided into two groups according to their diagnosis: 111 subjects with idiopathic PD and 111 control subjects. The neuropsychological examination was performed using the Frontal Assessment Battery, the copy of the Rey-Osterrieth complex figure and the digit test for determining frontal functioning. RESULTS AND CONCLUSIONS: We prove the existence of a frontal dysfunction that is characterised by impaired working memory, with visuospatial and executive dysfunction, which suggests greater involvement of the dorsolateral prefrontal cortex and cingulate. According to our findings, because working memory and visuospatial functioning are correlated to the motor status and the time elapsed since the onset of the disease, they could share the same underlying neuroanatomical foundations--the nigrostriatal denervation. This is not the case of executive function, which was not found to be related to the characteristics of the disease under study.

Cognition Disorders↗

PubMed1978

A soluble lipid.protein complex from bovine adrenal medulla chromaffin granules.

A unique soluble lipoprotein has been isolated from aqueous lysates of bovine adrenal medulla chromaffin granules by DEAE-cellulose chromatography and gel filtration. Chloroform/methanol extracts of this complex contain sphingomyelin, lecithin, and cholesterol. Gel filtration in aqueous media indicate an approximate molecular weight of 900,000 for the complex. Incubation with sodium dodecyl sulfate causes dissociation to a low molecular weight polypeptide; prolonged treatment with guanidine HCl does not promote dissociation at all. Amino acid analysis revealed a high content of hydrophobic amino acids. Analysis of the tryptic fingerprint indicates that a single type of polypeptide chain is present. The complex appears to contain approximately five copies of polypeptide per aggregate.

Adrenal Medulla↗

PubMed1993

High-level expression of secreted proteins from cells adapted to serum-free suspension culture.

We have developed a host cell/vector system based on the use of adenovirus-transformed cells and a promoter, designated GBMT, capable of being activated by the Ela tumor antigen produced in these cells. GBMT-based vectors were constructed with hygromycin phosphotransferase and murine dihydrofolate reductase as selective markers. We demonstrate their utility in two adenovirus-transformed cell lines, human kidney 293 and Syrian hamster AV12-664. Further, we describe methods and conditions for the direct adaptation of isolated recombinant clones to serum-free suspension growth conditions. For exemplary purposes, we describe the generation of stable recombinant 293 cell lines with single-copy integrated vectors secreting the highly complex clotting factor human protein C at levels as high as 20 mg/l in serum-free suspension culture. In addition, using the AV12-664 cell line with GBMT and direct dominant selection of the dhfr gene, we have isolated clones secreting a tissue plasminogen activator derivative at levels of about 40 mg/l under serum-free suspension conditions. The distinct advantages of this vector/host cell system are 1) the direct selection of stable clones expressing relatively high levels of recombinant protein, eliminating the need for the tedious stepwise gene amplification process and 2) the direct adaptation to serum-free suspension culture.

Adaptation, Physiological↗

PubMed1995

Analysis of genetic changes underlying local recurrence of prostate carcinoma during androgen deprivation therapy.

The molecular mechanisms and genetic changes that lead to the progression of prostate cancer during endocrine therapy are poorly characterized. Here, paired specimens from both untreated primary tumors and from local recurrences were collected from 10 prostate cancer patients treated by conventional androgen deprivation therapy. The genetic progression of the tumors was studied by using interphase fluorescence in situ hybridization and chromosome-specific probes. Six primary tumors (60%) and all ten recurrent tumors were aneuploid by fluorescence in situ hybridization. The recurrent tumors also showed a high degree of chromosome copy number variability from one cell to another. Increased copy number of chromosome X was particularly common in the recurrent tumors. In addition, specific high level amplification of the androgen receptor (AR) gene (Xq12) was detected in three highly aneuploid recurrent tumors. Our findings suggest that hormone-refractory prostate cancers are genetically very complex and show intratumor genetic heterogeneity. Increased copy number of chromosome X and the amplification of the androgen receptor (AR) gene may confer proliferative advantage during androgen deprivation and thus contribute to the development of recurrence.

Androgen Antagonists↗

PubMed2005

DNA-based classification and sequence heterogeneities in the 16S rRNA genes of Lactobacillus casei/paracasei and related species.

The sequence differences within the 16S rRNA genes of Lactobacillus casei/paracasei and related species, Lactobacillus zeae and Lactobacillus rhamnosus, were investigated. Thirty-seven strains of mostly human or cheese origin were grouped by restriction endonuclease analysis (REA) of the total chromosomal DNA and by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rRNA gene fragments. REA verified that all strains were genomically unique and singled out three major clusters, one L. rhamnosus-cluster and two clusters containing L. paracasei strains. The groups obtained by TTGE corresponded with one exception to the REA-clusters. In the TTGE clustering all L. paracasei strains formed one general group with one TTGE-band in common, and this group was sub-divided into five subgroups due to the presence of more than one TTGE-band in four of the subgroups. The occurrence of multiple TTGE-bands was investigated by amplifying and cloning of the 16S rRNA genes from the strains showing this phenomenon, thereby 12 clones from each strain were sequenced, demonstrating polymorphisms in almost all the cases. Subjecting the clones displaying sequence variations to TTGE as well as sequencing of 16S rDNA revealed by ribotyping of the strains, verified the presence of polymorphisms within the 16S rRNA genes. The migration characteristic of amplified DNA from a single clone corresponded to a specific band in the TTGE-pattern of the strain from which the clone originated. Southern blot hybridisation with a 16S rDNA probe demonstrated the presence of at least five 16S rRNA genes in L. casei/paracasei. A higher degree of variable positions than previously reported was observed in the 16S rRNA gene fragments of the members in the complex. Sequence comparison between the 16S rRNA gene copies of L. casei (CCUG 21451T) and L. zeae (CCUG 35515T) demonstrated that the two species shared almost the same sequence in some copies while the others were more different. Our results provide one explanation for the difficulties in reaching clear-cut taxa within the L. casei/paracasei complex.

Base Sequence↗

PubMed2000

Mechanisms of Hox gene colinearity: transposition of the anterior Hoxb1 gene into the posterior HoxD complex.

Transposition of Hoxd genes to a more posterior (5') location within the HoxD complex suggested that colinearity in the expression of these genes was due, in part, to the existence of a silencing mechanism originating at the 5' end of the cluster and extending towards the 3' direction. To assess the strength and specificity of this repression, as well as to challenge available models on colinearity, we inserted a Hoxb1/lacZ transgene within the posterior HoxD complex, thereby reconstructing a cluster with a copy of the most anterior gene inserted at the most posterior position. Analysis of Hoxb1 expression after ectopic relocation revealed that Hoxb1-specific activity in the fourth rhombomere was totally abolished. Treatment with retinoic acid, or subsequent relocations toward more 3' positions in the HoxD complex, did not release this silencing in hindbrain cells. In contrast, however, early and anterior transgene expression in the mesoderm was unexpectedly not suppressed. Furthermore, the transgene induced a transient ectopic activation of the neighboring Hoxd13 gene, without affecting other genes of the complex. Such a local and transient break in colinearity was also observed after transposition of the Hoxd9/lacZ reporter gene, indicating that it may be a general property of these transgenes when transposed at an ectopic location. These results are discussed in the context of existing models, which account for colinear activation of vertebrate Hox genes.

Animals↗

PubMed1992

Handwriting and spelling: dyslexic children's abilities compared with children of the same chronological age and younger children of the same spelling level.

Handwriting speed and spelling were examined in a group of 10-year-old dyslexic children compared with children of the same age and with younger children of the same spelling level. The children wrote lists of words onto a digitizer pad in three different condition: a dictation, copying from a sheet on the desk and copying from a wall chart. The words ranged in complexity from simple monosyllabic phoneme to grapheme words to words needing orthographic and morphological information and non-words. There were differences in writing speed between the 10-year-olds and 8-year-olds in most conditions. There were no significant differences in speed of writing or pausing between the dyslexic children and the 10-year-olds. There was a difference in the number of errors in the spelling of non-words, the dyslexic children being inferior to both the other groups. The only difference between the dyslexic children and the 8-year-olds was in speed of writing in copying from the desk and in writing complex words. The performance of the dyslexic group was more similar to that of the 8-year-olds in the dictation but to the 10-year-olds in the copying conditions. Independent judges had no difficulty in identifying the 10-year-olds' writing but confused that of the 8-year-olds with the dyslexic children's. It is proposed that the dyslexic children had automatised movement patterns linked to spelling equivalent to their same age peers but that these patterns were built on accumulated inaccuracies in both letter formation and spelling.

Achievement↗

PubMed1997

CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper-inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to -11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA-repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY-DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10(-9) M and 5 x 10(-9) M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 microM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.

Adenosine Triphosphatases↗

PubMed2004

Processing of complex stimuli and natural scenes in the auditory cortex.

Neuronal responses in auditory cortex show a fascinating mixture of characteristics that span the range from almost perfect copies of physical aspects of the stimuli to extremely complex context-dependent responses. Fast, highly stimulus-specific adaptation and slower plastic mechanisms work together to constantly adjust neuronal response properties to the statistics of the auditory scene. Evidence with converging implications suggests that the neuronal activity in primary auditory cortex represents sounds in terms of auditory objects rather than in terms of invariant acoustic features.

Action Potentials↗

PubMed1992

Mapping of the mouse ornithine decarboxylase-related sequence family.

A family of DNA sequences homologous to the mRNA encoding ornithine decarboxylase (ODC) and comprising approximately 12 members in the mouse genome has been analyzed genetically. The inheritance of variant DNA restriction fragments detected by ODC cDNA probes on Southern blots of DNA from inbred strain mice was determined in six sets of recombinant inbred (RI) mouse strains. The distributions of these variations among the RI strains were then compared with the RI strain distribution patterns (SDPs) of previously mapped loci. This allowed the identification of nine independent ODC-related loci, of which eight could be localized to specific regions of the mouse genome: Odc-rs1 near Lamb2 on Chromosome (Chr) 1; Odc-rs2 near Psp on Chr 2; Odc-rs5, a complex locus comprising at least 5-7 copies of the ODC sequence, associated with Igk on Chr 6; Odc-rs6 between Abpa and Tam-1 on proximal Chr 7; Odc-rs7 near Hbb on distal Chr 7; Odc-rs12 near Agt and Emv-2 on distal Chr 8; Odc-rs8 associated with the Igh complex on Chr 12; and Odc-rs9 near Otf-3f on Chr 14. The ODC-related sequence family thus comprises a set of genomically dispersed "marker" loci, and alleles for several of these loci can be analyzed simultaneously in DNA from mice or cell lines. DNA from mice of 70 inbred strains has been characterized for alleles at all nine Odc-rs loci.

Alleles↗

PubMed2003

Inactivation of the chloroplast ATP synthase gamma subunit results in high non-photochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana.

The nuclear atpC1 gene encoding the gamma subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the gamma subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase alpha and beta subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.

Actins↗

PubMed1983

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Bacillus subtilis↗

PubMed2006

Consistence of miniexon polymerase chain reaction-restriction fragment length polymorphism and single-copy gene sequence analyses in discriminating Leishmania genotypes.

Recently, a new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay had been developed using the miniexon sequences for genotyping Leishmania isolates. We had used this method for rapid diagnosis and genotyping of visceral and cutaneous leishmaniasis with the combination of microcapillary cultivation. In this study, we have evaluated this approach by examining genomic DNAs from 47 independent isolates, which were grouped into 19 genotypes of Leishmania subgenus complexes by sequence polymorphism of single-copy genes. Results obtained provide miniexon RFLP configurations specific to Leishmania enriettii, Leishmania tarentolae, and Leishmania gerbilli for the first time. Altogether, 92% of the results from miniexon PCR-RFLP are in agreement with those based on the sequence database of single-copy genes from the same isolates. The miniexon PCR-RFLP method is simple, sensitive, and specific method useful for routine diagnosis of different Leishmania.

Animals↗