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Trains of sleep sequences are indices of learning capacity in rats.

In previous work dealing with the identification of four sleep sequences (SS-->W, SS-->PS, SS-->TS-->W and SS-->TS-->PS) in the baseline session of adult male Wistar rats [Mandile P, Vescia S, Montagnese P, Romano F, Giuditta A. Characterization of transition sleep episodes in baseline EEG recordings of adults rats, Physiol Behav 1996;60:1435-1439], we have shown that those containing an intervening episode of transition sleep (TS) strongly correlate with the number of avoidances scored the following day [Vescia S, Mandile P, Montagnese P, Romano F, Cataldo G, Cotugno M, Giuditta A. Baseline transition sleep and associated sleep episodes are related to the learning ability of rats, Physiol Behav 1996;60:1513-152]. More recently, clusters of sleep sequences (trains) separated by waking intervals longer than 60 s have been identified in the baseline session of the same rats [Piscopo S, Mandile P, Montagnese P, Cotugno M, Giuditta A, Vescia S. Identification of trains of sleep sequences in adult rats, Behav Brain Res, this volume], and distinguished in homogeneous or mixed trains according to the presence of a single sleep sequence or more than one sequence. Mixed trains have been further separated into trains containing the SS-->TS-->W sequence (+TSW trains) and trains lacking it (-TSW trains). Analysis of the distribution of variables of baseline trains (and of their sleep sequences and components) among fast learning (FL), slow learning (SL), or non-learning (NL) rats, indicates that variables of +TSW trains prevail in FL rats, while variables of -TSW trains prevail in NL rats. In addition, variables of +TSW trains correlate with the number of avoidances of the training session, while variables of -TSW trains do not significantly correlate, or show inverse correlations. Interestingly, sleep sequences such as SS-->W or SS-->TS-->W show direct or inverse correlations with avoidances depending on whether they are included in +TSW trains or in -TSW trains. The data are interpreted to suggest that the outcome of brain operations performed during a sleep sequence may selectively condition the appearance of later sequences within a time interval shorter than a given threshold. An analogous mechanism may be responsible for the aggregation of sleep components in sleep sequences.

Analysis of Variance↗

Organization and orientation of a human T cell receptor delta chain V gene segment that suggests an inversion mechanism is utilized in its rearrangement.

A human T cell receptor (TcR) delta chain variable region segment, V delta 3, has been mapped 2 kilobases 3' to the constant (C delta) gene. The V delta 3 gene segment is in an inverted transcriptional orientation to the diversity regions (D delta), joining regions (J delta) and C delta gene segments. Analyses of TcR delta cDNA clone, KT041, showed that V delta 3 is linked to D delta 1, D delta 2, J delta 3 and C delta and that this message encodes a potentially functional TcR delta chain. These findings indicate that a functional TcR delta gene can be constructed through a chromosomal inversion, suggesting that in human, chromosomal inversion is one of the important rearrangement mechanisms.

Base Sequence↗

In vivo analysis of the plasmid pAM beta 1 resolution system.

The promiscuous plasmid pAM beta 1 from Gram-positive bacteria encodes a resolution system which differs from that of Tn3 in that (i) it requires a histone-like protein and an unusual resolvase-DNA interaction to promote recombination and (ii) it mediates in vivo DNA inversion in plasmid substrates. In this in vivo analysis, the pAM beta 1 resolution site is narrowed down to a 99 bp segment, the strand exchange is mapped within 10 bp and the serine residue at position 10 of the resolvase is shown to be essential for enzyme activity. In addition, data showing that the resolution system does not promote DNA inversion in the Bacillus subtilis chromosome are presented. Implications of this observation are discussed.

Bacillus subtilis↗

A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements.

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

DNA Restriction-Modification Enzymes↗

Fast method for longitudinal relaxation time and water content mapping of the human brain on a clinical MR scanner.

BACKGROUND: Longitudinal relaxation time (T(1)) map generation from human brain slices renders possible the in vivo follow-up of the changes in T(1) values during the course of several pathologies such as stroke, multiple sclerosis, traumatic brain injury etc. T(1) values can be converted to water contents, thus brain oedema reducing therapy can be non-invasively evaluated. The purpose of the study was to work out a fast and simple MRI method to obtain T(1) and water maps of the human brain. METHOD: The T(1) values of Gadolinium solutions with different concentrations were determined by means of MRI methods at a clinical MR scanner operating at 1 Tesla. In order to validate these measurements, T(1) values of the same Gadolinium solutions were also quantified with a relaxometer operating at the same field strength. T(1) and water maps from the brains of healthy volunteers were obtained with an inversion prepared spoiled gradient echo sequence (turbo-FLASH). FINDINGS: The T(1) values of Gadolinium solutions measured with the relaxometer showed a strong correlation (r > 0.999) with those determined with MRI sequences on the whole body scanner. The fastest MRI method to produce T(1) and consequent water maps from human brain was the inversion prepared turbo-FLASH sequence. CONCLUSIONS: The implemented turbo-FLASH method can produce T(1) and water map of a single virtual brain slice within 2 minutes. However, brain tissue containing haemorrhage should be excluded from the measurement due to the large influence of excessive haemoglobin concentration on longitudinal relaxation. The proposed method is available on most of the MR scanners, thus T(1) and water mapping of human brain can be routinely performed.

Adult↗

A prospective assessment of breath-hold fast spin echo and inversion recovery fast spin echo techniques for detection and characterization of focal hepatic lesions.

The purpose of this study was to prospectively assess two breath-hold T(2)-weighted fast spin-echo sequences and two breath-hold inversion recovery fast spin-echo sequences to determine their relative ability to detect and characterize focal hepatic lesions. Fourteen patients with a total of nineteen proven focal hepatic lesions were imaged with two breath-hold T(2)-weighted (T2W) fast spin echo sequences (HASTE TE = 66 and HASTE TE = 120), two breath-hold inversion recovery fast spin echo sequences (IRFSE TE = 64 and IRFSE TE = 95), and a nonbreath-hold T(2)-weighted fast-spin echo sequence (FSE TE = 96-120). Contrast-to-noise ratios (CNRs) were measured for all proven lesions on all sequences. Both IRFSE sequences and the HASTE sequence with TE = 66 showed an improvement in lesion-liver and liver-spleen CNRs compared to the nonbreath-hold T2W sequence. The mean difference in CNR between benign and malignant lesions was largest for the HASTE TE = 120 sequence. These preliminary results suggest that a breath-hold IRFSE sequence (TE = 64 or 95) has an equal ability to detect focal hepatic lesions as a nonbreath-hold T2W FSE sequence (TE = 96-120). The HASTE TE = 120 showed the greatest ability to discriminate between benign and malignant lesions.

Adult↗

Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157.

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.

Amino Acid Sequence↗

Carrier detection and prenatal diagnosis of hemophilia A.

The aim of this study was to establish a simple, rapid carrier detection and prenatal diagnosis system for hemophilia A. Intron 22 inversion in FVIII gene was directly examined by long-distance polymerase chain reaction. Polymorphisms of factor VIII intragenic restriction fragment length polymorphism of Bcl I, short tandem repeat (STR) within intron 13 and 22, and extragenic DXS 52 (St 14) variable number tandem repeats (VNTR) loci were assessed by hereditary linkage analysis. The diagnostic rates for these loci were 47.6% (intron 22 inversion), 27.8% (Bcl I, 28.6% and 29.4% (STR within intron 13 and 22), and 81.3% (DXS52), respectively. The overall diagnostic rate in 21 families was 94.7%. The diagnosis in hemophilia A patients or carriers can be made if intron 22 inversion is present. The intragenic and extragenic loci hereditary linkage analysis could be used to establish the diagnosis in intron 22 inversion-negative patients.

Base Sequence↗

Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin.

An Escherichia coli chromosomally coded factor termed FIS (Factor for Inversion Stimulation) stimulates the Cin protein-mediated, site-specific DNA inversion system of bacteriophage P1 more than 500-fold. We have purified FIS and the recombinase Cin, and studied the inversion reaction in vitro. DNA footprinting studies with DNase I showed that Cin specifically binds to the recombination site, called cix. FIS does not bind to cix sites but does bind to a recombinational enhancer sequence that is required in cis for efficient recombination. FIS also binds specifically to sequences outside the enhancer, as well as to sequences unrelated to Cin inversion. On the basis of these data, we discuss the possibility of additional functions for FIS in E. coli.

Base Sequence↗

MR cholangiography in children with autosomal recessive polycystic kidney disease.

BACKGROUND: Magnetic resonance cholangiography (MRC) is a relatively new, non-invasive imaging technique of the biliary tree that has shown good correlation with endoscopic retrograde cholangiopancreatography. The liver manifestation of autosomal recessive polycystic kidney disease (ARPKD) is congenital hepatic fibrosis (CHF). CHF may be accompanied by Caroli's disease, which is characterised by a non-obstructive dilation of the intrahepatic bile ducts. OBJECTIVE: A prospective study was conducted to determine the presence and extent of Caroli's disease in children with ARPKD. MATERIALS AND METHODS: Seven children with ARPKD aged from 3.0 to 10. 1 years were examined. CHF was confirmed in all biopsied cases (5 of 7). All children had been followed by repeated abdominal US examinations for many years. The MR examination included a morphological imaging study using a T2-weighted turbo spin-echo sequence and a heavily T2-weighted inversion-recovery turbo spin-echo sequence with three-dimensional maximum intensity projection (MIP) reconstructions for MRC. RESULTS: The diagnosis of Caroli's disease could be made in one case by US; in two other children Caroli's disease was suspected, but the differentiation from hepatic cysts was not possible. By MRC, Caroli's disease could be diagnosed in three of seven children. Furthermore, MRC with MIP reconstructions demonstrated the extent of the disease by showing the entire biliary tree from different angles. CONCLUSIONS: MRC is a valuable method to establish the diagnosis and demonstrate the extent of Caroli's disease.

Bile Ducts, Intrahepatic↗

Charge hopping in DNA.

The efficiency of charge migration through stacked Watson-Crick base pairs is analyzed for coherent hole motion interrupted by localization on guanine (G) bases. Our analysis rests on recent experiments, which demonstrate the competition of hole hopping transitions between nearest neighbor G bases and a chemical reaction of the cation G(+) with water. In addition, it has been assumed that the presence of units with several adjacent stacked G bases on the same strand leads to the additional vibronic relaxation process (G(+)G...G) --> (GG...G)(+). The latter may also compete with the hole transfer from (G(+)G...G) to a single G site, depending on the relative positions of energy levels for G(+) and (G(+)G...G). A hopping model is proposed to take the competition of these three rate steps into account. It is shown that the model includes two important limits. One corresponds to the situation where the charge relaxation inside a multiple guanine unit is faster than hopping. In this case hopping is terminated by several adjacent G bases located on the same strand, as has been observed for the GGG triple. In the opposite, slow relaxation limit the GG...G unit allows a hole to migrate further in accord with experiments on strand cleavage exploiting GG pairs. We demonstrate that for base pair sequences with only the GGG triple, the fast relaxation limit of our model yields practically the same sequence- and distance dependencies as measurements, without invoking adjustable parameters. For sequences with a certain number of repeating adenine:thymine pairs between neighboring G bases, our analysis predicts that the hole transfer efficiency varies in inverse proportion to the sequence length for short sequences, with change to slow exponential decay for longer sequences. Calculations performed within the slow relaxation limit enable us to specify parameters that provide a reasonable fit of our numerical results to the hole migration efficiency deduced from experiments with sequences containing GG pairs. The relation of the results obtained to other theoretical and experimental studies of charge transfer in DNA is discussed. We propose experiments to gain a deeper insight into complicated kinetics of charge-transfer hopping in DNA.

Base Pairing↗

Repetitive DNA sequences near three human beta-type globin genes.

Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes. A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene. Each contains a homologous Alu I site. Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene. A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.

Base Sequence↗

The mouse T complex gene Tsga2, encoding polypeptides located in the sperm tail and anterior acrosome, maps to a locus associated with sperm motility and sperm-egg interaction abnormalities.

Previous studies of sperm from mice heterozygous for a t haplotype (t) and heterospecific combinations of the t complex identified two tightly linked genetic factors responsible for t/t male sterility related to expression of the flagellar waveform aberration, curlicue. Dnahc8, an axonemal dynein heavy chain gene, is a strong candidate for the proximal factor, Ccua, but the identity of the distal factor, Ccub, is unknown. In the present study, we employ motility assays of sperm from males heterozygous for t and novel heterospecific combinations of the t complex to demonstrate that Ccub is a composite of at least two synergic elements, Ccub1, positioned within a genomic interval spanning approximately 0.6 Mb immediately distal to Dnahc8, and Ccub2, situated in a region approximately 4-7 Mb distal to Ccub1. We also show that Tsga2, a testis-restricted gene, fulfills many of the prerequisites required to make it a strong candidate for Ccub1. These include: 1) its location within the aforementioned genomic interval; 2) a highly reduced level of testis expression by its heterospecific allele relative to the level of expression of its t allele; 3) determination that TSGA2(t) carries numerous nonsynonymous mutations in residues otherwise highly conserved in all known orthologous proteins; 4) the detection of major TSGA2 polypeptides in sperm protein extracts; and 5) the apparent distribution of these polypeptides in major sperm tail structures. Surprisingly, these TSGA2 isoforms appear to localize in the vicinity of the anterior acrosome, as well, suggesting that Tsga2 may also play a role in sperm-egg interaction. Finally, our results indicate that a TSGA2 polypeptide with apparent similarities to the smaller of the two sperm isoforms is expressed by epididymal cells.

Acrosome↗

Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal enterococcus faecium population.

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.

Animal Husbandry↗

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.).

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.

3' Untranslated Regions↗

[FLAIR images of mild head trauma with transient amnesia].

A newly advanced MRI pulse sequence, the FLAIR (fluid-attenuated inversion recovery) imaging, in which a long TE spin echo sequence is used with suppression of the CSF with an inversion pulse, displays the CSF space as a no signal intensity area. We examined 45 cases of mild head trauma with posttraumatic amnesia by FLAIR images and could detect some findings which could not be detected by CT scan and conventional MR images. These findings could be detected in many patients with long post-traumatic amnesia (over 2 hours), but they could not be detected in patients with short posttraumatic amnesia (within 30 mins). These findings existed surrounding lateral ventricles and we classified them into 3 types: type 1 is anterior horn of lateral ventricle, type 2 is the base of frontal lobe, type 3 is cerebral deep white matter. Some of them were examined again by FLAIR images a month later, and these findings had disappeared. We suspect that these lesions were brain edema or mild contusion without hemorrhage.

Adolescent↗