The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3'' untranslated regions between man and chicken.

Dystrophin is a very large muscle protein (approximately 400 kd) the deficiency of which is responsible for Duchenne muscular dystrophy. Its function is unknown at present. In order to know whether different domains of the protein are differentially conserved during evolution, we have cloned and sequenced the chicken dystrophin cDNA. The protein coding sequence has almost the same size as in man. The N-terminal region that resembles the actin binding domain of alpha actinin, as well as the large spectrin like domain show 80% and 75% conservation respectively between chicken and man. In contrast, the C-terminal region shows 95% identity over 627 aa suggesting that it is an important region of interaction with other proteins. Comparison of the amino acid sequence of this C-terminal region to other protein sequences shows only marginally significant similarities. Finally we have found a striking conservation of three segments of the 3' untranslated sequence (85% homology over a total of 920 nt) between chicken and man. These also appear to be conserved in other mammals. This high conservation is not linked to open reading frames.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

A somatic component to myocardial infarction.

Sixty two patients were randomised to be seen by osteopathic physicians for palpation of the thoracic paravertebral soft tissue, T1-T8. Twenty five patients had clinically confirmed acute myocardial infarction. Of the remainder, 22 without known cardiovascular disease served as controls and 15 were placed in an excluded group because of diagnosed cardiovascular disease other than myocardial infarction. Observations were described in predetermined standard terminology. The control group was found to have a low incidence of palpable changes throughout the thoracic dorsum, and these changes were uniformly distributed from T1 to T8. Examination of the group with myocardial infarction disclosed a significantly higher incidence of soft tissue changes (increased firmness, warmth, ropiness, oedematous changes, heavy musculature), confined almost entirely to the upper four thoracic levels. The 15 patients who were excluded from the experimental group because they had various cardiovascular diseases other than myocardial infarction also showed significantly different changes on palpation compared with the group with myocardial infarction. These findings suggest that myocardial infarction is accompanied by characteristic paravertebral soft tissue changes which are readily detected by palpation.     

Repetitive satellite-like sequences are present within or upstream from 3 avian protein-coding genes.

Peculiar DNA sequences made up by the tandem repetition of a 5 bp unit have been identified within or upstream from three avian protein-coding genes. One sequence is located within an intron of the chicken "ovalbumin-X" gene with 5'-TCTCC-3' as basic repeat unit (36 repeats). Another sequence made of 27 repeats of a 5'-GGAAG-3' basic unit is found 2500 base pairs upstream from the promoter of the chicken ovotransferrin (conalbumin) gene. A related but different sequence is present in the corresponding region of the ovotransferrin gene in the pheasant, with 5'-GGAAA-3' as the basic unit (55 repeats). These three satellite-like elements are thus characterized by a total assymetry in base distribution, with purines restricted to one strand, and pyrimidines to the other. Two of the basic repeat units can be derived from the third one (GGAAA) by a single base pair change. These related sequences are found repeated in three avian genomes, at degrees which vary both with the sequence type and the genome type. Evolution of tandemly repeated sequences (including satellites) is in general studied by analysing randomly picked elements. The presence of conserved protein-coding regions neighbouring satellite-like sequences allow to follow their evolution at a single locus, as exemplified by the striking comparison of the pheasant and chicken sequences upstream from the ovotransferrin gene.     

c-fos expression in the amygdala:In vivo antisense modulation and role in anxiety

Summary 1. The amygdaloid complex is a key structure in mechanisms of fear and anxiety. Expression of the immediate-early gene c-fos has been reported in the central nucleus of the amygdala following various stressors, but the functional role of this phenomenon has remained unknown.2. c-fos expression was observed in the central nucleus when rats were subjected to a pharmacologically validated animal model of anxiety, the Vogel conflict test, but not after mere exposure to the test apparatus. Bilateral amygdala injection of a 15-mer phosphorothioate c-fos antisense oligodeoxynucleotide prior to testing blocked conflict-induced c-fos expression and had behavioral effects similar to those of established antianxiety drugs.3. Separate experiments determined that antisense treatment did not affect conflict behavior by acting on shock thresholds or drinking motivation.4. These findings provide evidence that neuronal activation and c-fos induction in the amygdala may be of importance for mechanisms of fear and anxiety.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

The ovalbumin gene family: complete sequence and structure of the Y gene.

The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from the comparison with the ovalbumin gene sequence and from the map previously established by electron microscopy analysis. During evolution of the Y gene, selective pressure has operated to retain a sequence coding for an ovalbumin-like protein. The location of splice junctions, the length of protein coding exons and the reading phase are as in the ovalbumin gene. The overall homology between the Y and ovalbumin protein coding sequences is 72.6% (resulting in a 58% homology for the amino acid sequences). A significantly high number of base changes within coding sequences are present in clusters, which appear in several cases to be correlated with the occurrence of direct repeats. The 3' untranslated sequences of the Y and ovalbumin mRNAs have diverged much more, and the Y sequence contains a peculiar U(T) rich region. Corresponding introns of the ovalbumin and Y genes differ extensively both in sequence and in length. They share however characteristic biases in their base distribution.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.