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Coordinate regulation of metabolic enzyme encoding genes during cardiac development and following carvedilol therapy in spontaneously hypertensive rats.

Fuel substrate utilization is highly regulated during cardiac development and with the onset of cardiac hypertrophy. Glucose and lactate are the predominant fuel substrates utilized during cardiac development. Postnatally, a switch occurs so that fatty acids become the chief energy substrate in the nonfed adult mammalian heart. A reversion back towards fetal energy metabolism occurs with the development of cardiac hypertrophy. To evaluate the role of this substrate preference switch in the development of cardiac hypertrophy, the molecular regulation directing these switches is being explored. Thus, we have begun by defining the temporal expression patterns of genes encoding key rate-controlling enzymes directing major fuel substrate metabolism during cardiac development, with pressure-overload-induced cardiac hypertrophy, and following antihypertensive therapy in spontaneously hypertensive rats. The genes encoding the fatty acid and adult enriched rate-controlling glycolytic enzymes are expressed at low levels in the fetal and neonatal rat heart. The genes encoding these enzymes are significantly and coordinately upregulated (> or = 70%) in adult rat hearts compared to the fetal expression patterns. A reciprocal and coordinate downregulation (> or = 40% reduction) of the fatty acid and adult enriched glycolytic enzyme encoding genes are observed with the induction of pressure-overload-induced hypertrophy in spontaneously hypertensive rats compared to Wistar-Furth normotensive control rats. Antihypertensive therapy with carvedilol, a vasodilating alpha- and beta-adrenoreceptor antagonist, attenuates this reversion of the metabolic gene expression pattern towards fetal levels compared to placebo-treated littermate controls. This coordinate developmental and hypertrophy-induced regulation of genes that encode enzymes controlling both fatty acid and glycolytic catabolic pathways in the heart implicates potential mutual/overlapping regulatory signaling proteins within their gene regulatory programs. These gene regulatory pathways need to be identified and modulated in order to characterize the functional role of fuel substrate metabolism in cardiac development and with the induction of cardiac hypertrophy.

Acyl-CoA Dehydrogenase↗

Assessing the performance of neural encoding models in the presence of noise.

An analytical method is introduced for evaluating the performance of neural encoding models. The method addresses a critical question that arises during the course of the development and validation of encoding models: is a given model near optimal in terms of its accuracy in predicting the stimulus-elicited responses of a neural system, or can the predictive accuracy be improved significantly by further model development? The evaluation method is based on a derivation of the minimum mean-square error between actual responses and modeled responses. It is formulated as a comparison between the mean-square error of the candidate model and the theoretical minimum mean-square error attainable through an optimal model for the system. However, no a priori information about the nature of the optimal model is required. The theoretically minimum error is determined solely from the coherence function between pairs of system responses to repeated presentations of the same dynamic stimulus. Thus, the performance of the candidate model is judged against the performance of an optimal model rather than against that of an arbitrarily assumed model. Using this method. we evaluated a linear model for neural encoding by mechanosensory cells in the cricket cercal system. At low stimulus intensities, the best-fit linear model of encoding by single cells was found to be nearly optimal, even though the coherence between stimulus-response pairs (a commonly used measure of system linearity) was low. In this low-stimulus-intensity regime, the mean square error of the linear model was on the order of the power of the cell responses. In contrast, at higher stimulus intensities the linear model was not an accurate representation of neural encoding. even though the stimulus-response coherence was substantially higher than in the low-intensity regime.

Action Potentials↗

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana.

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 and sdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.

Amino Acid Sequence↗

Cloning and sequence analysis of cDNA encoding thiamin-binding proteins from sesame seeds.

The amino acid sequences of the large polypeptides of thiamin-binding proteins (TBPs) from sesame (Sesamum indicum L.) seeds (STBP-I, -II and -III) were analyzed. The large polypeptides of STBP-I, -II and -III had the same amino acid sequences as did their small polypeptides. The peptide sequence information obtained from STBPs was used to synthesize DNA primers for amplification of the gene(s) encoding STBPs. A 200-bp fragment was amplified from cDNA synthesized from RNA from sesame seeds 4 weeks after flowering. The 200-bp fragment was used to clone full-length cDNA(s) encoding STBP(s) with RACE techniques. A 644-bp fragment was amplified, cloned and sequenced. The cDNA was a full-length clone encoding STBP(s). It contained an open reading frame, which defined a 143-residue polypeptide. The identified small and large polypeptide sequences of STBPs exactly matched the sequence encoded within the cDNA clone. These results indicated that the small and large polypeptides of STBPs were encoded on the mRNA as a single large proprotein precursor and that the final mature forms were generated by post-translational processing in the same manner as the other 2S albumins of plant seeds.

Journal Article↗

Encoding and decoding nonverbal cues of emotion.

Subjects ("senders") encoded six emotions twice, first via facial expressions and second via tone of voice. These expressions were recorded and presented for decoding to the senders and an additional group of judges. Results were as follows: (a) the ability to encode and the ability to decode both visual and auditory cues were significantly related; (b) the relationship between encoding and decoding cues of the same emotion appeared low or negative; (c) the ability to decode visual cues was significantly related to the ability to decode auditory cues, but the correlations among encoding (and decoding) scores on different emotions were low; (d) females were slightly better encoders, and significantly better decoders, than males; (e) acquaintance between sender and judge improved decoding scores among males but not among females; (f) auditory decoding scores were higher than visual decoding scores, particularly among males; (g) auditory decoding scores were relatively high if sender and judge were of the same sex, while visual decoding scores were relatively high if sender and judge were of opposite sexes; (h) decoding scores varied according to channel of communication and type of emotion transmitted.

Anger↗

Self-perpetuating development of encoding biases.

The process of encoding new information involves the imposition of preexisting interpretive categories on newly encountered stimuli, even if the categories do not match perfectly those stimuli. We hypothesized that such encoding of stimuli as supportive of preexisting encoding dispositions may become a source of a perceiver's subjective experiences that support these dispositions. Through this nonconsciously operating mechanism, encoding rules may gradually develop in a self-perpetuating manner, even in the absence of any objectively supportive evidence. Results demonstrated this self-perpetuating process in three studies involving different stimulus materials and experimental tasks (matrix-scanning paradigm and two "intuitive judgment" tasks). The self-perpetuating development of encoding biases is discussed as one of the elementary mechanisms involved in the development of interpretive categories and other individually differentiated cognitive dispositions.

Adult↗

Effects of divided attention on encoding and retrieval processes in human memory: further support for an asymmetry.

Despite a tradition in cognitive psychology that views encoding and retrieval processes in human memory as being similar, F. I. M. Craik, R. Govoni, M. Naveh-Benjamin and N. D. Anderson (1996) have recently shown that notable differences exist between the 2 when divided-attention manipulations are used. In this article, the authors further examined this asymmetry by using several manipulations that changed task demands at encoding and retrieval. The authors also used a secondary-task methodology that allowed a microlevel analysis of the secondary-task costs associated with encoding and retrieval. The results illustrated the resiliency of retrieval processes to manipulations involving different task demands. They also indicated different loci of attention demands at encoding and retrieval. The authors contend that whereas encoding processes are controlled, retrieval processes are obligatory but do require attentional resources for their execution.

Attention↗

Encoding of geometric and featural spatial information by goldfish (Carassius auratus).

Goldfish (Carassius auratus) were trained in different place-finding tasks as a means of analyzing their ability to encode the geometric and the featural properties of the environment. Results showed that goldfish could encode and use both geometric and featural information to navigate. Goldfish trained in a maplike, or relational, procedure encoded both types of information in a single representation. In contrast, fish trained in a directly cued procedure developed 2 independent and competing strategies. These results suggest that the geometric properties of the spatial arrangement and discrete landmarks are sensitive to encoding in a maplike or relational system, whereas different sources of spatial information are encoded in a single and flexible representation of the environment.

Animals↗

Identification of a protein encoded by the vpu gene of HIV-1.

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.

Acquired Immunodeficiency Syndrome↗

Putative transcription activator with alternative isoforms encoded by human ZFX gene.

The ZFY gene in the sex-determining region of the human Y chromosome encodes a protein with 13 zinc fingers, and may determine whether an embryo develops as a male or female. ZFX, a related gene on the human X chromosome, may also function in sex determination; it encodes a protein with a very similar zinc-finger domain and escapes X inactivation. ZFY and ZFX diverged from a common ancestral gene before the radiation of placental mammals, and retain a similar genomic organization. Analysis of complementary DNAs from the mouse Y-chromosomal homologues of ZFY indicates that these genes encode probable transcription activators. Here, we report that ZFX encodes a protein composed of a highly acidic amino-terminal domain, a basic putative nuclear-localization signal, and a carboxy-terminal zinc-finger domain. This combination of features, also found in the ZFY gene product, is typical of transcription activators. Alternative splicing generates ZFX transcripts encoding isoforms of 575 and 804 amino acids. These ZFX protein isoforms differ in the length of their acidic domains and may be functionally distinct.

Amino Acid Sequence↗

Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.

Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.

Amino Acid Metabolism, Inborn Errors↗

Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4-13). These dominant forms of MED (EDM1-3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1-3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24-p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.

Adult↗

Biomolecular screening with encoded porous-silicon photonic crystals.

Strategies to encode or label small particles or beads for use in high-throughput screening and bioassay applications focus on either spatially differentiated, on-chip arrays or random distributions of encoded beads. Attempts to encode large numbers of polymeric, metallic or glass beads in random arrays or in fluid suspension have used a variety of entities to provide coded elements (bits)--fluorescent molecules, molecules with specific vibrational signatures, quantum dots, or discrete metallic layers. Here we report a method for optically encoding micrometre-sized nanostructured particles of porous silicon. We generate multilayered porous films in crystalline silicon using a periodic electrochemical etch. This results in photonic crystals with well-resolved and narrow optical reflectivity features, whose wavelengths are determined by the etching parameters. Millions of possible codes can be prepared this way. Micrometre-sized particles are then produced by ultrasonic fracture, mechanical grinding or by lithographic means. A simple antibody-based bioassay using fluorescently tagged proteins demonstrates the encoding strategy in biologically relevant media.

Animals↗

Attenuating the growth of tumors by intratumoral administration of DNA encoding Pseudomonas exotoxin via cationic liposomes.

A gene therapy approach was taken to inhibit tumor growth by transfecting tumor cells with a plasmid encoding a truncated but active form of Pseudomonas exotoxin A (PE), using cationic lipids as the transfection reagent. Cells transfected with this plasmid express PE intracellularly and undergo apoptosis. Transfection was optimized in vitro using two cationic lipids, DOGS and DOSPER. A ratio of between 1:4 and 1:10 (wt/wt) was found to be optimal for DOSPER, and the ratio 1:4 was used for the in vivo study when a smaller injection volume was desired. Estimating the activity of the PE-encoding plasmid was done both directly, by counting cells in vitro after transfection, and by using a cytotoxicity assay, and indirectly, by cotransfecting the plasmid with a plasmid carrying a reporter beta-galactosidase gene and observing a reduction in beta-galactosidase activity with increasing amounts of the PE-encoding plasmid. The cotransfection method was found to be very sensitive, and showed transfection of cells even with 1-2 ng of the PE-encoding plasmid per 10(5) cells. Complexes of the PE-encoding plasmid together with cationic lipid were injected into tumor xenografts in athymic nude mice. The tumor growth of transfected tumors was attenuated compared with control untreated tumors or tumors transfected with a nontoxin-expressing vector. These results indicate the potential of such a treatment for attenuating solid tumor growth in vivo.

ADP Ribose Transferases↗

Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope.

The potency of DNA vaccines may be affected by the efficiency of intracellular processing and MHC class I presentation of encoded antigens. Since a single-chain trimer (SCT) composed of peptide, beta2-microglobulin (beta2m), and MHC class I heavy chain has been shown to bypass antigen processing and lead to stable presentation of peptides, we investigated the efficacy of a DNA vaccine encoding a SCT composed of an immunodominant CTL epitope of human papillomavirus type 16 (HPV-16) E6 antigen, beta2m, and H-2Kb MHC class I heavy chain (pIRES-E6-beta2m-Kb). Transfection of 293 cells with pIRES-E6-beta2m-Kb can bypass antigen processing and lead to stable presentation of E6 peptide. Furthermore, C57BL/6 mice vaccinated with pIRES-E6-beta2m-Kb exhibited significantly increased E6 peptide-specific CD8+ T-cell immune responses compared to mice vaccinated with DNA encoding wild-type E6. Most importantly, 100% of mice vaccinated with pIRES-E6-beta2m-Kb DNA were protected against a lethal challenge of E6-expressing TC-1 tumor cells. In contrast, all mice vaccinated with wild-type E6 DNA or control plasmid DNA grew tumors. Our data indicate that a DNA vaccine encoding a SCT can lead to stable enhanced MHC class I presentation of encoded antigenic peptide and may be useful for improving DNA vaccine potency to control tumors or infectious diseases.

Animals↗

A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia.

Modern therapy for pediatric acute lymphoblastic leukemia (ALL) is based on the principle of risk stratification. One of the most important laboratory features used to accurately risk stratify patients is the presence of specific chromosomal translocation within the leukemic blasts. In this paper, we describe a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the accurate, sensitive, and rapid identification of chimeric transcripts encoded by the major risk-stratifying translocations of pediatric ALL. This assay will identify both the CML- and ALL-type BCR-ABL transcripts encoded by the t(9;22), all described variants of the E2A-PBX1 transcripts encoded by the t(1;19), the MLL-AF4 transcripts encoded by the t(4;11), and all variants of TEL-AML1 encoded by the t(12;21). In addition, we have developed a reverse dot-blot detection system as an alternative to traditional post-PCR Southern blot analysis. Application of this combined assay to the analysis of 70 leukemic samples and five cell lines resulted in a complete concordance between this multiplex assay and individual PCR reactions. The characteristics of the multiplex assay suggest that its application to routine clinical screening will significantly improve the ability of clinical laboratories to accurate risk stratify pediatric ALL patients.

Child↗

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

Amino Acid Sequence↗

A high-stringency proteomics concept aimed for generation of antibodies specific for cDNA-encoded proteins.

A novel dual bacterial expression system, designed for high-throughput generation of antibodies specific for cDNA-encoded proteins, is presented. The concept involves parallel expression of cDNA-encoded proteins, in two vector systems, as fusions with two different tags, both enabling single-step affinity purification under denaturing conditions. One of the fusion tags includes a portion with documented immunopotentiating effect to stimulate antibody production, and the generated fusion proteins are used to elicit antibodies. The second fusion protein is used in an immobilized form as an affinity ligand to enrich, from the generated antisera, antibodies with selective reactivity to the cDNA-encoded part. To evaluate the system, five cDNA clones from a mouse testis cDNA library were expressed, and antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The five antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the five antibodies gave specific staining in Western-blot screening of various cell types and tissue homogenates. When the same five cDNAs were processed and analysed using a single-vector method, antibodies with a more non-specific staining were generated. We thus conclude that the presented dual-vector method offers a highly stringent strategy for generation of monospecific polyclonal antibodies.

Animals↗