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PubMed2003

Processing abstract auditory features in the human auditory cortex.

Using electric and magnetic brain responses we tested whether violations of an abstract auditory regularity are processed in auditory cortex and whether abstract auditory regularities are retained for at least 10 s. The mismatch negativity (MMN) event-related potential and its magnetic counterpart (MMNm) were recorded to infrequent tone pairs of descending pitch (the second tone having a lower frequency than the first one) embedded in a sequence of tone pairs of ascending pitch, whereas the absolute frequency of both ascending and descending tone pairs varied on seven levels. Results showed that the dominant generators of the electromagnetic activity elicited by violations of the pitch-ascension rule lie within auditory cortex. We also found that the memory representation of pitch-ascension is retained for at least 10 s. When short trains of ascending-pitched tone pairs were followed by a silent period of 8-12 s, descending-pitched probe tone pairs elicited the MMN component when a single reminding pair with ascending pitch was presented before the probe. The reactivating effect of the reminder was similar to what has been previously shown for concrete auditory regularities, such as the constancy of tone pitch. The present results support the view that auditory cortical functions can process sensory and categorical information in a similar manner.

Acoustic Stimulation↗

PubMed2005

A preliminary phylogeny of the Pentatomomorpha (Hemiptera: Heteroptera) based on nuclear 18S rDNA and mitochondrial DNA sequences.

Pentatomomorpha is the second suborder in size only to Cimicomorpha in Heteroptera. However, the phylogenetic relationships among members of the suborder are not well established. Sequences from partial nuclear ribosomal 18S gene and mitochondrial COX1 gene were analyzed separately and in combination to generate a preliminary molecular phylogeny of Pentatomomorpha based on 40 species representing 17 putative families. Analyses of the combined sequence data provided a better-resolved and more robust hypothesis of Pentatomomorpha phylogeny than did separate analyses of the individual genes. The phylogenies were mostly congruent with morphological studies. Results strongly supported the monophyly of the infraorder Pentatomomorpha, and the placement of Aradoidea as sister to Trichophora. The monophyletic Trichophora was grouped into two major lineages, one being the superfamily Pentatomoidea, and the other comprising Lygaeoidea, Coreoidea, and Pyrrhocoroidea. The analysis of the ML and ME trees of combined dataset supported the monophyletic Pentatomoidea. In all analysis the Pyrrhocoroidea was polyphyletic; the monophyletic Lygaeoidea was supported only in the analysis of ME tree, and Coreoidea was polyphyletic except in the MP tree of combined dataset. The molecular and morphylogical data both indicated that the family Coreoidae should be revised subsequently. Our phylogenetic results suggested that the COX1 segment alone might not be an optimal molecular marker for the phylogeny of Pentatomomorpha.

Animals↗

PubMed1999

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.

Animals↗

PubMed2000

The genetic basis for cytopathogenicity of pestiviruses.

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by their effects on tissue culture cells. Identification of cp bovine viral diarrhea virus (BVDV) has been frequently reported since antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system, only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed a second way for the development of a cp pestivirus which is based on the introduction of a set of point mutations within the NS2 gene.

Animals↗

PubMed1986

Factors modulating the effects of teleost telencephalon ablation on retention, relearning, and extinction of instrumental avoidance behavior.

Two experiments on the nature of avoidance behavior in the goldfish are reported. In the first experiment, a master/yoked design was used to determine the degree to which Pavlovian conditioning would generate acquisition of a shuttle response by goldfish. Results indicated that the mere sequence of signal-shock pairings experienced by the avoidance animals is not in this situation sufficient to explain the development of the instrumental avoidance responding. The second experiment was conducted to study the effects of three variables on the retention and extinction of the avoidance response. First, unilateral lesions of the telencephalon produced a transient deficit from which the subjects recovered to normal levels. Retention of the avoidance response after bilateral telencephalon ablation was impaired and never fully recovered with additional training. Second, this deficit was reduced under higher intensities of electric shock. Third, overtraining on the task before the ablation had only a nonsignificant enhancing effect on retention levels. Extinction was always poorer following ablation, independent of the type of ablation, intensity of shock, degree of overtraining, and terminal retention level of performance. The results are discussed in terms of various theories of telencephalic function in fish.

Animals↗

PubMed1997

Sequence identity of the terminal redundancies on the minus-strand DNA template is necessary but not sufficient for the template switch during hepadnavirus plus-strand DNA synthesis.

The template for hepadnavirus plus-strand DNA synthesis is a terminally redundant minus-strand DNA. An intramolecular template switch during plus-strand DNA synthesis, which permits plus-strand DNA elongation, has been proposed to be facilitated by this terminal redundancy, which is 7 to 9 nucleotides long. The aim of this study was to determine whether the presence of identical copies of the redundancy on the minus-strand DNA template was necessary and/or sufficient for the template switch and at what position(s) within the redundancy the switch occurs for duck hepatitis B virus. When dinucleotide insertions were placed within the copy of the redundancy at the 3' end of the minus-strand DNA template, novel sequences were copied into plus-strand DNA. The generation of these novel sequences could be explained by complete copying of the redundancy at the 5' end of the minus-strand DNA template followed by a template switch and then extension from a mismatched 3' terminus. In a second set of experiments, it was found that when one copy of the redundancy had either three or five nucleotides replaced the template switch was inhibited. When the identical, albeit mutant, sequences were restored in both copies of the redundancy, template switching was not necessarily restored. Our results indicate that the terminal redundancy on the minus-strand DNA template is necessary but not sufficient for template switching.

Animals↗

PubMedDate not supplied

Ultrafast contrast-enhanced three-dimensional MR angiography: state of the art.

Ultrafast breath-hold contrast material-enhanced magnetic resonance (MR) angiography can be performed with a flexible imaging sequence. With the current generation of high-speed imaging gradients, it is possible to achieve sequence repetition times of 4 msec or less. These repetition times make it possible to obtain high-resolution (512 x 512 x 64) images in under 30 seconds. Applications of this versatile technique include imaging of aortic dissection, thoracic and abdominal aortic aneurysm, pulmonary embolus, carotid stenosis, and peripheral vascular disease. The administration of contrast material must be tailored to the vascular anatomy under examination to avoid venous enhancement. The rapid data acquisition times can be used to image multiple temporal phases or multiple locations. With this technique and administration of a T1-shortening contrast agent, high-quality MR angiography can be routinely performed in a variety of vascular regions (eg, thoracic and abdominal aorta, pulmonary arteries, carotid arteries, lower extremities).

Aortic Aneurysm↗

PubMed2003

Intracellular renin and the nature of intracrine enzymes.

Recently, the binding of renin and prorenin to cellular receptors with the subsequent generation of second messengers and the production of physiological effects has been demonstrated. In addition, the internalization of prorenin by target cells has been associated with increased cellular synthesis of angiotensin and cardiac pathology. Also, a renin transcript lacking the sequences encoding a secretory signal has been reported, and this transcript appears to produce a renin that acts in the cell that synthesized it. Some years ago, we coined the term intracrine for a peptide hormone or factor that acts in the intracellular space either after internalization or retention in its cell of synthesis. Thus defined, a wide variety of peptides display intracrine functionality, including hormones, growth factors, transcription factors, and enzymes. For example, considerable evidence indicates that angiotensin II is an intracrine. Also, general principles of intracrine functionality have been developed. Thus, recent evidence demonstrates that the prorenin/renin molecule is an intracrine enzyme. Here, the actions of intracrine enzymes (angiogenin, phosphoglucose isomerase, phospholipase A2, granzyme A and B, thioredoxin, platelet-derived endothelial growth factor, and serine protease inhibitors) are reviewed. The relation of prorenin/renin to other intracrine enzymes, and to intracrines in general, is discussed.

Animals↗

PubMed2000

[Clinical and molecular characterization of autosomal recessive chronic granulomatous disease caused by p47-phox deficiency].

BACKGROUND: The cytosolic protein p47-phox (phagocyte oxidase) is one of the essential components of the superoxide generating system in phagocytes and its defect causes approximately 30% of the chronic granulomatous disease (CGD) cases. AIM: Two patients were studied, belonging to the same family, without a consanguinous background, in which deficiency or absence of superoxide generation was found together with recurrent and severe infections in one case and benign infections in the second. METHODS: The presence of gp91-, p67- and p47-phox in patients and controls was determined by Western Blot analysis of granulocytes. Sequencing of PCR amplified DNA was performed by an enzymatic method. RESULTS: Western Blot analysis showed normal expression of gp91 and p67 and absence of p47-phox. The molecular genetic study demonstrated a homocygotic dinucleotide GT (GT) deletion at the beginning of exon 2 of the p47-phox gene. The same mutation has been found in European, American and Japanese patients. CONCLUSIONS: The molecular characterization of this pathology done for the first time in Chile is important for diagnostic classification, patient prognosis, and adequate genetic advice and a possible future therapy.

Adolescent↗

PubMed1991

[Focused extracorporeal pyrotherapy. Initial experimental results].

The objective of this study was to develop an apparatus allowing the generation of a high temperature (exceeding 80 degrees C) in a precise focus (20 mm x 2 mm) by means of extracorporeal elastic waves. The treatment time at high temperatures is brief and administered in sequences of 4 to 7 seconds. In vitro studies on blocks of polyurethane demonstrated melting of the plastic at the focal point. Studies on plastic spheres introduced into the bladder of the pig demonstrated melting of the sphere without any alteration in the tissues in the wave path. Studies of cellular viability of bladder carcinoma cultures demonstrated a significant difference after 48 hours between the non-treated control group and the group of cells submitted to high temperatures. This technique, called Pyrotherapy, should be promising if the preliminary results are confirmed.

Animals↗

PubMed1997

[Technical aspects of fast, ultra-fast MR imaging].

Technical aspects of fast, ultra-fast MR imaging are described. Firstly, mechanism of steady state MR signals, generated by rf pulses with short repetition times, are focused, and described that the steady state signal contains echo signals that have different history. Imaging pulse sequence utilizing the steady state signals is referred to as Gradient Recalled Echo (GRE) sequence. Secondly, in order to give general description for echo train imaging sequences, concept of k-space and k-space trajectory is introduced. The concept is useful to realize how echo train imaging sequences such as echo planar imaging and fast spin echo imaging collect raw data efficiently.

Humans↗

PubMed1998

A novel neuropeptide precursor gene is expressed in the terrestrial snail central nervous system by a group of neurons that control mating behavior.

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior.

Amino Acid Sequence↗

PubMed2002

Binding of oligonucleotides to a viral hairpin forming RNA triplexes with parallel GGC triplets.

Infrared and UV spectroscopies have been used to study the assembly of a hairpin nucleotide sequence (nucleotides 3-30) of the 5' non-coding region of the hepatitis C virus RNA (5'-GGCGGGGAUUAUCCCCGCUGUGAGGCGG-3') with a RNA 20mer ligand (5'-CCGCCUCACAAAGGUGGGGU-3') in the presence of magnesium ion and spermidine. The resulting complex involves two helical structural domains: the first one is an intermolecular duplex stem at the bottom of the target hairpin and the second one is a parallel triplex generated by the intramolecular hairpin duplex and the ligand. Infrared spectroscopy shows that N-type sugars are exclusively present in the complex. This is the first case of formation of a RNA parallel triplex with purine motif and shows that this type of targeting RNA strands to viral RNA duplexes can be used as an alternative to antisense oligonucleotides or ribozymes.

Base Sequence↗

PubMed1993

Molecular basis of an adult form of Sandhoff disease: substitution of glutamine for arginine at position 505 of the beta-chain of beta-hexosaminidase results in a labile enzyme.

Sandhoff disease is a lysosomal storage disorder characterized by accumulation of GM2 ganglioside due to mutations in the beta-chain of beta-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the beta-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the beta-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G-->A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the beta-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for DdeI, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with DdeI resulted in complete digestion at the mutation site, demonstrating that the second allele was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of beta-hexosaminidase. These results show that the patient's is a genetic compound, and that the lability of beta-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition.

Amino Acid Sequence↗

PubMed2005

Novel sequence for generating glycopolymer tethered on a membrane surface.

Cell surface carbohydrates, usually binding with other biomacromolecules (such as lipids and proteins), are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation, and inflammation. Synthetic carbohydrate-based polymers, so-called glycopolymers, are emerging as important well-defined tools for investigating carbohydrate-based biological processes and for simulating various functions of carbohydrates. In this study, a novel two-step sequence for the generation of a glycopolymer layer tethered on a polypropylene microporous membrane is described. First, a UV-induced graft polymerization of 2-aminoethyl methacrylate hydrochloride (AEMA) was carried out on the membrane to generate an amino-functionalized surface, and the effects of polymerization factors (monomer/initiator concentration and UV irradiation time) on the grafting density were studied. Second, sugar moieties were bound with the grafted functional layer to form glycopolymer by the reaction between the amino groups on the membrane surface and carbohydrate lactones. Chemical analysis by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy combined with surface morphology observation by scanning electron microscopy confirmed the graft polymerization of AEMA and the formation of glycopolymer. The decreases of water contact angle and protein adsorption on the membrane revealed the enhancement of hydrophilicity and protein resistance due to the typical characteristics of the glycopolymer tethered on the surface. These results indicated that the novel sequence reported in this work is a facile process to form glycopolymer-modified surfaces.

Cell Membrane↗

PubMed2001

Single amino acid substitutions and deletions that alter the G protein coupling properties of the V2 vasopressin receptor identified in yeast by receptor random mutagenesis.

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.

Amino Acid Sequence↗

PubMed1991

Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

Animals↗

PubMed1980

HgiAI: a restriction endonuclease from Herpetosiphon giganteus HP1023.

A new class II restriction endonuclease, HgiAI has been partially purified from Herpetosiphon giganteus HP1023. The enzyme activity has been characterized and shown to recognize the family of related hexanucleotide sequences (Formula: see text) where the second and fifth nucleotide pairs are A:T pairs in either orientation. Cleavage occurs as shown, to give DNA fragments with 3'-terminal tetranucleotide extensions. The recognition sites of the enzymes SacI and SstI (Formula: see text) form a subset of the recognition site of HgiAI. One of the four possible tetranucleotide 3'-extensions (cohesive ends), generated by HgiAI is identical with those generated by SacI and SstI, another is identical with that of PstI. HgiAI should be useful for molecular cloning.

Base Sequence↗