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Degradation of MYCN oncoprotein by the ubiquitin system.

Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins as a class. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the MYCN gene. The oncoproteins were synthesized in vitro by transcription of an MYCN cDNA, and translation of the resulting single species of mRNA in the presence of 35S-methionine. Degradation of labeled proteins was monitored in a cell-free system derived from rabbit reticulocytes. ATP stimulated the degradation of the MYCN protein more than 10 fold. ATP-dependent degradation was completely inhibited by neutralizing antibody directed against E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, ATP-dependent degradation in E1-depleted lysate could be restored by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the ATP-dependent degradation of MYCN oncoproteins in vitro, and that these proteins possess signals that target them for rapid turnover by this proteolytic pathway. Although MYCN in neuroblastomas is activated primarily by amplification, it may be activated by other mechanisms in aggressive tumors with a single copy of MYCN. Mutations in protein coding sequence that alter the signals for recognition by protein degradation systems represent a potential mechanism by which oncogene activation might occur.

Animals↗

[Role of nonspecific complex formation in protein-nucleic acid recognition].

Combinatory mathematical model of equilibrium binding of recognising proteins to DNA has been considered. The expression for the ratio of specific and unspecific complex as the function of a number of nucleotides in DNA, a number of centers of recognition, concentration of recognising molecules and a code number have been obtained. The analysis of obtained expression for the repressor--operator system showed that binary code would preferable than quartic one. The specificity of binding corresponding to experimental results have been observed by the number of centers more than twenty, in the narrow interval of the energy value and by high limitation on the number of combinations of error centers.

Base Sequence↗

Helper T-cell recognition of HIV-1 Tat synthetic peptides.

The regulatory proteins coded by the human immunodeficiency virus, (HIV)-1 genome are expressed by the infected cells before the initiation of the synthesis of structural proteins and thus immune response directed against these proteins could destroy infected cells before the release of infectious virions. The evaluation of T-lymphocyte responses toward Tat, one of the main HIV-1 regulatory proteins, is therefore of interest. We selected a group of HIV-infected patients with retained response to the recall antigen purified protein derivative and tested their CD4+ helper T-cell response toward recombinant Tat and toward 12 soluble synthetic partially overlapping 15-16-mer Tat peptides in a proliferation assay. Three peptides (amino acids 17-32, 33-48, and 65-80) were significantly recognized by the helper T-cells from infected individuals but not by the nine HIV-1-negative control persons. Nine of the 14 patients (64%) responded to at least one of these Tat peptides. Of the identified immunodominant peptides containing T-cell epitopes, one (aa 65-80) was recognized in association with human leukocyte antigens DR-2 allele, while the others appeared to be promiscuous and were equally recognized in association with several DR molecules. The identified immunogenic peptides were analyzed for the predicted presence of T-cell antigenic sites by several algorithms and positive correlation was detected for each peptide. Our results thus indicate that Tat protein can induce a cell-mediated immune response and identify three peptides containing T-cell epitopes that may be of importance in vaccine development.

Alleles↗

New developments in speech pattern element hearing aids for the profoundly deaf.

Two new developments in speech pattern processing hearing aids will be described. The first development is the use of compound speech pattern coding. Speech information which is invisible to the lipreader was encoded in terms of three acoustic speech factors; the voice fundamental frequency pattern, coded as a sinusoid, the presence of aperiodic excitation, coded as a low-frequency noise, and the wide-band amplitude envelope, coded by amplitude modulation of the sinusoid and noise signals. Each element of the compound stimulus was individually matched in frequency and intensity to the listener's receptive range. Audio-visual speech receptive assessments in five profoundly hearing-impaired listeners were performed to examine the contributions of adding voiceless and amplitude information to the voice fundamental frequency pattern, and to compare these codings to amplified speech. In both consonant recognition and connected discourse tracking (CDT), all five subjects showed an advantage from the addition of amplitude information to the fundamental frequency pattern. In consonant identification, all five subjects showed further improvements in performance when voiceless speech excitation was additionally encoded together with amplitude information, but this effect was not found in CDT. The addition of voiceless information to voice fundamental frequency information did not improve performance in the absence of amplitude information. Three of the subjects performed significantly better in at least one of the compound speech pattern conditions than with amplified speech, while the other two performed similarly with amplified speech and the best compound speech pattern condition. The three speech pattern elements encoded here may represent a near-optimal basis for an acoustic aid to lipreading for this group of listeners. The second development is the use of a trained multi-layer-perceptron (MLP) pattern classification algorithm as the basis for a robust real-time voice fundamental frequency extractor. This algorithm runs on a low-power digital signal processor which can be incorporated in a wearable hearing aid. Aided lipreading for speech in noise was assessed in the same five profoundly hearing-impaired listeners to compare the benefits of conventional hearing aids with those of an aid which provided MLP-based fundamental frequency information together with speech+noise amplitude information. The MLP-based pattern element aid gave significantly better performance in the reception of consonantal voicing contrasts from speech in pink noise than that achieved with conventional amplification and consequently, it also gave better overall performance in audio-visual consonant identification.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗

[Digital image processing: fundamental techniques and applications in biological research].

Digital image processing represents a relatively new but very dynamic domain. Its importance is still growing due to the rapid development in related computer equipment. In this paper basics concepts are introduced and a survey of problems and methods is presented. Starting with techniques used for the capture of data, it continues with techniques applied to coding new data, image enhancement and pattern recognition. Special attention is given to pattern recognition techniques which provide the basics for classification of data according to established criteria. Finally, two applications are presented; the reconstruction of 3-D volumes from serial cutting and a survey of chlorophyll concentration on Lake Aculeo using remote images obtained by the LANDSAT satellite.

Analog-Digital Conversion↗

[Value of a predictive model of ambulatory blood pressure integrating physical activity].

OBJECTIVE: To determine how much of the variations of blood pressure during a 24 hour period could be accounted for by a change in activity and establish a predictive model. MATERIALS AND METHODS: Twenty three healthy subjects (mean age 25 +/- 2 years) were studied. The BP, heart rate (HR), and time of measure (T) were recorded by ambulatory BP monitoring using Spacelabs (4 measures per hour). At each measure the subject noted in a diary the degree of activity on a six level semi-quantitative scale. DATA ANALYSIS: A model was constructed using an analysis of covariance. Different parameters were added in succession to reach a model of the type P: P0 + A + beta + (HR-HR0) + H, were P = predicted systolic pressure, P0 = mean systolic BP over the 24 hours. A variation in systolic BP for activity level, beta = the slope of the regression between systolic BP and HR during activity A, and HR0 the mean HR during this activity. RESULTS: 1) In order to test the model, the values measured in one subject were compared to the predicted values from the model in 22 others. The procedure was then repeated for the other subjects. This common model predicted 41 +/- 21% of fluctuations in BP of the subject analysed with a range of 0 to 66%. 2) In order to refine the individual model two subjects were explored 7 times over 24 h of non consecutive days. The measures of the last recording were compared to the predicted values from the application of the model to the six preceding recordings. The model then predicted 81% and 66% of the BP values of the test day. The mean of the 24 hour individual difference over a one hour period between the measures and its predicted value by the model was 0.13 +/- 4.8 mmHg, and -0.75 +/- 7.7 mmHg. CONCLUSION: This study expresses in a quantitative fashion the importance of the level of activity in the evaluation of the level of ambulatory BP. The introduction of this method of quantification and analysis seems logical in therapeutic trial. The difference in the predictions by the model for some subjects poses the problem of uniform coding of activities and that of the recognition of other events such as stress and dreaming in sleep.

Adult↗

[Y chromosome and male infertility].

Spermatogenesis is a complex physiological process characterized by an orderly proliferation and differentiation of germ cell types, from diploid spermatogonial stem cells to haploid spermatids, and after spermiogenesis to spermatozoa. It is known that male reproductive capacity is deficient in about one half of infertile couples. Effective treatment is available only for a small fraction of these infertile men. In most cases, the cause of male infertility is unknown. Aetiologically, male infertility may result from genetic and non-genetic causes. Among the genetic factors known to disturb spermatogenesis, chromosomal aberrations, involving autosomes and/or sex chromosomes, are well known. However, molecular genetic causes of idiopathic male infertility have been accumulating in the last years, predominantly for the Y chromosome. Localization of genes that control spermatogenesis on Yq11 was first proposed, on cytogenetic evidence, by Tiepolo and Zuffardi in 1976. Since then, many subsequent reports using molecular genetics methods have supported the initial proposal by detecting submicroscopic interstitial deletions on Yq11, present in patients with idiophatic azoospermia and a cytogenetically normal Y chromosome. Recently, four spermatogenesis candidate Y-linked genes (or gene families), RBM, DAZ, SPGY and TSPY have been cloned and characterized. In the euchromatic Y chromosome long arm three intervals--AZFa, AZFb and AZFc--were also defined. All of them may contain genes necessary for normal spermatogenesis. All the four genes have a testis-specific expression and three of them (RBM, DAZ and SPGY) code for ribonucleoproteins with a single RNA recognition motif. Candidate genes with in AZFb are some members of the RBM gene family and within AZFc are the DAZ and SPGY gene family. While AZFc deletions are associated with azoospermia or with severe oligoteratoasthenozoospermia, microdeletions involving AZFa or AZFb cause azoospermia only.

Chromosome Mapping↗

Place coding of vowel formants for cochlear implant patients.

Four multiple-channel cochlear implant patients were tested with synthesized versions of the words "hid, head, had, hud, hod, hood" containing 1, 2, or 3 formants, and with a natural 2-formant version of the same words. The formant frequencies were encoded in terms of the positions of electrical stimulation in the cochlea. Loudness, duration, and fundamental frequency were kept fixed within the synthetic stimulus sets. The average recognition scores were 47%, 61%, 62%, and 79% for the synthesized 1-, 2-, and 3-format vowels and the natural vowels, respectively. These scores showed that the place coding of the first and second formant frequencies accounted for a large part of the vowel recognition of cochlear implant patients using these coding schemes. The recognition of the natural stimuli was significantly higher than recognition of the synthetic stimuli, indicating that extra cues such as loudness, duration, and fundamental frequency contributed to recognition of the spoken words.

Attention↗

Unfocused spatial attention underlies the crowding effect in indirect form vision.

We studied mechanisms underlying the crowding effect in indirect form vision by measuring recognition contrast sensitivity of a character with flankers to the left and right. Attentional and featural contributions to the effect can be separated by a new paradigm that distinguishes pattern location errors from pattern recognition errors and further by manipulating the focusing of spatial attention through a positional cue, appearing 150 ms before the target. Measurements were on the horizontal meridian, at 1, 2, and 4 deg eccentricity, and a range of flankers' distances were used. Our results show that in normal indirect viewing, the impairment of character recognition by crowding is--in particular at intermediate flanker distances--caused to a large part by spatially imprecise focusing of attention. In contrast, the enhancement of performance by a transient positional cue seems mediated through a separate attentional mechanism such that attentional locus and focus are controlled independently. Our results furthermore lend psychophysical support to a separate coding of the what and where in pattern recognition.

Attention↗

Recognition of own-race and other-race caricatures: implications for models of face recognition.

Valentine's (Valentine T. Q J Exp Psychol 1991;43A:161-204) face recognition framework supports both a norm-based coding (NBC) and an exemplar-only, absolute coding, model (ABC). According to NBC; (1) faces are represented in terms of deviations from a prototype or norm; (2) caricatures are effective because they exaggerate this norm deviation information; and (3) other-race faces are coded relative to the (only available) own-race norm. Therefore NBC predicts that, for European subjects, caricatures of Chinese faces made by distorting differences from the European norm would be more effective than caricatures made relative to the Chinese norm. According to ABC; (1) faces are encoded as absolute values on a set of shared dimensions with the norm playing no role in recognition; (2) caricatures are effective because they minimise exemplar density and (3) the dimensions of face-space are inappropriate for other-race faces leaving them relatively densely clustered. ABC predicts that all faces would be recognised more accurately when caricatured against their own-race norm. We tested European subjects' identification of European and Chinese faces, caricatured against both race norms. The ABC model's prediction was supported. European faces were also rated as more distinctive and recognised more easily than Chinese faces. However, the own-race recognition bias held even when the races were equated for distinctiveness which suggests that the ABC model may not provide a complete account of race effects in recognition.

Facies↗

A range/domain approximation error-based approach for fractal image compression.

Fractals can be an effective approach for several applications other than image coding and transmission: database indexing, texture mapping, and even pattern recognition problems such as writer authentication. However, fractal-based algorithms are strongly asymmetric because, in spite of the linearity of the decoding phase, the coding process is much more time consuming. Many different solutions have been proposed for this problem, but there is not yet a standard for fractal coding. This paper proposes a method to reduce the complexity of the image coding phase by classifying the blocks according to an approximation error measure. It is formally shown that postponing range\slash domain comparisons with respect to a preset block, it is possible to reduce drastically the amount of operations needed to encode each range. The proposed method has been compared with three other fractal coding methods, showing under which circumstances it performs better in terms of both bit rate and/or computing time.

Algorithms↗

Comparison of different speech coding strategies using a disability-based inventory and speech perception tests in quiet and in noise.

OBJECTIVE: Intraindividual comparison of two cochlear implant speech coding strategies implemented in the Nucleus 24M system (SPEAK versus ACE). Reasons for subjective preference were evaluated using a combination of speech perception scores and a disability-based inventory. STUDY DESIGN: Cross-over study with two groups of cochlear implant subjects, assigned to first receive ACE or SPEAK strategy. SETTING: Cochlear implant program. SUBJECTS: Twelve postlingually deaf adults using a Nucleus 24M cochlear implant system. INTERVENTION: Subjects consecutively used the two different speech coding strategies and completed speech perception tests in quiet and in noise and a disability-based inventory (Abbreviated Profile of Hearing Aid Benefit). MAIN OUTCOME MEASURES: Individual differences between the two different speech coding strategies and the relation with subjective strategy preference. RESULTS: The ACE strategy produced somewhat better speech recognition scores in quiet and in noise, although the difference in the scores in noise did not reach the 95% level of significance. The first coding strategy chosen did not affect the results or subjective preference. Preference according to the APHAB results were not a priori in conformity with the speech recognition scores. CONCLUSION: Most subjects preferred the ACE strategy. Subjective preference was in agreement with the APHAB results in three subjects, in agreement with speech recognition in two subjects, and in agreement with both in seven subjects. The present results support the use of both speech recognition tests and questionnaires to evaluate different speech coding strategies.

Adult↗

Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice.

We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.

Amino Acid Sequence↗

Predicting internal exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames.

A new method which predicts internal exon sequences in human DNA has been developed. The method is based on a splice site prediction algorithm that uses the linear discriminant function to combine information about significant triplet frequencies of various functional parts of splice site regions and preferences of oligonucleotides in protein coding and intron regions. The accuracy of our splice site recognition function is 97% for donor splice sites and 96% for acceptor splice sites. For exon prediction, we combine in a discriminant function the characteristics describing the 5'-intron region, donor splice site, coding region, acceptor splice site and 3'-intron region for each open reading frame flanked by GT and AG base pairs. The accuracy of precise internal exon recognition on a test set of 451 exon and 246693 pseudoexon sequences is 77% with a specificity of 79%. The recognition quality computed at the level of individual nucleotides is 89% for exon sequences and 98% for intron sequences. This corresponds to a correlation coefficient for exon prediction of 0.87. The precision of this approach is better than other methods and has been tested on a larger data set. We have also developed a means for predicting exon-exon junctions in cDNA sequences, which can be useful for selecting optimal PCR primers.

Algorithms↗

Experimental studies on the origin of the genetic code and the process of protein synthesis: a review update.

This article is an update of our earlier review (Lacey and Mullins, 1983) in this journal on the origin of the genetic code and the process of protein synthesis. It is our intent to discuss only experimental evidence published since then although there is the necessity to mention the old enough to place the new in context. We do not include theoretical nor hypothetical treatments of the code or protein synthesis. Relevant data regarding the evolution of tRNAs and the recognition of tRNAs by aminoacyl-tRNA-synthetases are discussed. Our present belief is that the code arose based on a core of early assignments which were made on a physico-chemical and anticodonic basis and this was expanded with new assignments later. These late assignments do not necessarily show an amino acid-anticodon relatedness. In spite of the fact that most data suggest a code origin based on amino acid-anticodon relationships, some new data suggesting preferential binding of Arg to its codons are discussed. While information regarding coding is not increasing very rapidly, information regarding the basic chemistry of the process of protein synthesis has increased significantly, principally relating to aminoacylation of mono- and polyribonucleotides. Included in those studies are several which show stereoselective reactions of L-amino acids with nucleotides having D-sugars. Hydrophobic interactions definitely play a role in the preferences which have been observed.

5'-Nucleotidase↗

Performance of compressed analogue (CA) and continuous interleaved sampling (CIS) coding strategies for cochlear implants in quiet and noise.

Speech understanding with compressed analogue (CA) and continuous interleaved sampling (CIS) coding strategies for cochlear implants was compared in quiet and in noise at signal-to-noise ratios (SNRs) of 15, 10 and 5 dB. The speech recognition of three experienced users of the Ineraid cochlear implant (CA coding strategy) was assessed using a set of sentence, vowel and consonant tests. Three weeks after the fitting of a CIS processor, the tests were repeated with the new device. Speech recognition scores for the sentence and consonant tests tended to be higher with the CIS processor in no or little noise, but lower in the test situations with less favourable SNRs, when compared to the CA processor (average score differences for the consonant test: +7.8% correct at 15 dB SNR; -6.8% correct at 5 dB SNR; p = 0.05). Results for the vowel test were slightly lower on average for the CIS processing strategy at all SNRs. A possible explanation for the differences in performance between CIS and CA in the consonant and sentence tests at different SNRs is the generally higher free-field threshold associated with the CA coding strategy, which may act as a single-channel noise suppression.

Adolescent↗

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana.

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)(+)-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses. The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5' non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5' sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3' sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site. This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.

Amino Acid Sequence↗

Priming of T helper cells by antigen-activated B cells. B cell-primed Lyt-1+ helper cells are restricted to cooperate with B cells expressing the IgvH phenotype of the priming B cells.

Activated B cells isolated shortly after primary immunization of BALB/c donor mice with sheep erythrocytes (SRBC), were transferred to normal syngeneic recipients or to low-dose cyclophosphamide-pretreated syngeneic recipients. In pretreated recipients, the transfer of activated B cells, but not of T cells or macrophages, resulted in an augmented production of indirect plaque-forming cells in the primary immune response to SRBC but not to horse erythrocytes. It was shown in double-transfer experiments that T helper cells (Lyt-1+) had been stimulated by the transfer of antigen-activated B cells. Criss-cross double-transfer experiments using the mouse strains CB20 and BAB14 (congenic to BALB/c at the loci coding for the immunoglobulin heavy chain) indicate that those T helper cells are primed after recognition of B cell products that are encoded for by genes linked to the loci coding for the variable region of the immunoglobulin heavy chain (IgVH). The thus-primed Ig-dependent T helper cells (THIg) are adaptively restricted to cooperate with B cells that display IgVH-linked gene products similar to those that originally stimulated the THIg. These findings suggest that in the course of an immune response to T cell-dependent antigens, help for the production of specific IgG can be provided by THIg that have been primed and/or clonally expanded after recognition of IgVH-linked gene products by (e.g., complementary) T cell receptors.

Animals↗