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PubMed1981

Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological cross-reactivity with the rat liver enzyme.

The purification of mitochondrial carbamoyl phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) from small samples of human liver is described. The enzyme is composed of a single polypeptide of Mr 160 000 +/- 500 as shown by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents. The synthetase migrates in polyacrylamide gradient gels in the absence of detergents at a rate corresponding to a Mr of 165 000. Estimates of the molecular weight of the native enzyme by gel filtration and density gradient centrifugation yield a value of 178 000. The results indicate that the enzyme exists predominantly as monomeres. Amino acids composition, isoelectric point, stability, Km values and the ability to catalyze partial reactions have been measured and compared with known properties of carbamoyl phosphate synthetases from other sources. From the available data a high degree of evolutionary conservation of the ammonia-dependent synthetase is suggested. This is also supported by the demonstration of extensive immunological cross-reactivity between the human and rat enzymes.

Amino Acids↗
PubMed1990

Complementation of the Aspergillus nidulans arg B1 mutation by ornithine transcarbamylase cDNA from rat liver.

An Aspergillus nidulans strain which is deficient in ornithine transcarbamylase due to the arg B1 mutation was transformed with a plasmid containing the ornithine transcarbamylase cDNA from rat liver under the control of the amd S promoter. Stable transformants were obtained by selection on arginine free medium indicating complementation of the arg B mutation. Proof of expression of the rat enzyme in transformants was obtained by immunoprecipitation of all ornithine transcarbamylase activity from cell extracts with antibodies specific for the rat enzyme. The presence of catalytically active rat ornithine transcarbamylase in the transformants indicated that it is capable of being imported into mitochondria in A. nidulans, proteolytically processed and assembled into its homotrimeric form. In vitro uptake experiments using isolated A. nidulans mitochondria demonstrate that processing of the precursor of rat ornithine transcarbamylase occurs in two temporally separated steps as it does in rat liver mitochondria suggesting evolutionary conservation of the processing machinery. Up to 560 ng of active rat enzyme was produced per gm wet weight mycelia. Use of beta-D-alanine, an inducer of amd S, as sole N-source resulted in increased levels of active rat ornithine transcarbamylase relative to uninduced cultures.

Animals↗
PubMed1984

Glial fibrillary acidic protein and S-100 protein in pineal supportive cells: an electron microscopic study.

Pineal supportive cells in a teleost were shown to contain the glial fibrillary acidic (GFA) and S-100 proteins by electron microscopic immunocytochemistry. Both proteins are known to exhibit a high degree of evolutionary conservation. The pineal photoreceptor cells did not stain for these marker proteins. A relationship between supportive cells and macroglial elements is therefore implied.

Animals↗
PubMed1988

The binding of a monoclonal antibody reactive with pp60v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synaptic region.

A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.

Animals↗
PubMed1991

Promotion of neuronal survival in vitro by thermal proteins and poly(dicarboxylic)amino acids.

Evaluating molecules for their ability to promote survival and growth of neurons, we tested thermal proteins on cultures of dissociated fetal rat forebrain neurons. (Thermal proteins are polyamino acids formed when mixtures of amino acids with minimal proportions of glutamic or aspartic acid are heated.) Thermal proteins, added to low-density cultures in serum-free medium, stimulated neurite outgrowth and induced the formation of neuronal networks which survived for 6-10 days. Neurons in control cultures failed to grow and degenerated completely within 2-4 days. Effective concentrations (EC50) of thermal proteins ranged from 3 to 100 micrograms/ml. They were equally effective when present in the medium during the culture time or after precoating of the culture dishes. A single preparation which contained only aspartic and glutamic acid was effective, and similar survival promoting actions were then found for polyglutamic acid and mixed polyamino acids containing glutamic or aspartic acid. Thermal proteins and polyglutamic acid acted in a specific manner since, under the same experimental conditions, many control peptides, proteins and growth hormones failed to promote survival of neurons. Furthermore, their effects were antagonized by heparin, but not heparan sulfate nor chondroitin sulfate. These findings suggest that sequences of successive dicarboxylic amino acid residues are able to promote survival and neurite elongation of cultured neurons and that such sequences are responsible for the survival promoting action of thermal proteins. They invite the speculation that sequences of successive dicarboxylic amino acids, while occur in many proteins and show a high degree of evolutionary conservation, may have functional role in molecular recognition processes during neuronal development.

Amino Acid Sequence↗
PubMed1983

Affinity-purified antigen-specific products produced by T cells share epitopes recognized by heterologous antisera raised against several different antigen-specific products from T cells.

Heterologous antisera to murine or rat T-cell antigen-binding molecules (T-ABM) were raised in rabbits or sheep. The T-ABM used for immunization were purified by affinity for antigen and did not bear known immunoglobulin isotypes. T-ABM and anti-T-ABM were raised in three separate laboratories. Antisera to T-ABM were exchanged and tested for binding to T-ABM in three separate laboratories. Thus antisera to at least three distinct T-ABM were tested directly for binding to T-ABM or by adsorption of biological activity. Rabbit antisera to murine trinitrophenol (TNP)-specific T-ABM or rat AgB-specific T-ABM bound both murine or rat T-ABM, indicating evolutionary conservation of T-ABM. Similar results were found with sheep antisera to murine T-ABM. In addition, all heterologous anti-T-ABM antisera used bound murine T-ABM specific for TNP, 4-hydroxy-3-nitrophenyl acetate (NP), SRBC, or T-cell membrane proteins with similar structure. Thus, there is a commonality of antigenic determinants between various T-ABM and T-cell membrane homologues which may be T-cell surface receptors for foreign antigen.

Animals↗
PubMed1989

Islet amyloid polypeptide (IAPP):cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus.

We have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyteminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, racoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog). Thus, a species-specific structural motif in the putative amyloidogenic region of IAPP is associated with both amyloid formation and the development of age-related diabetes mellitus. This provides a new molecular clue to the pathogenesis of this disease.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells.

Among a library of monoclonal antibodies (mAbs) recognizing developmental markers in the chick embryo, mAb CC-3 was selected because of its differential immunostaining of mitotic cells. The intracellular distribution of the CC-3 antigen (CC-3a) throughout the cell cycle was visualized by immunolocalization. In interphase cells CC-3a resided in the nucleus and was arranged in distinct extranucleolar clusters. At prophase, the nuclear reactivity of CC-3a considerably increased and subsequently extended to the cytoplasm at metaphase. From metaphase through anaphase, most of the reactivity was associated with the mitotic apparatus. During cytokinesis CC-3a was detected in the mid-body and also in discrete speckles dispersed throughout the cytoplasm. The initial interphase pattern was then restored in the two daughter nuclei. Immunoblot analysis demonstrated that a 255-kDa phosphoprotein was present only in the interphase nucleus and that a complete new set of phosphoproteins accounted for the mitotic cell reactivity. The binding of CC-3 was dependent on the phosphorylation of its antigens. CC-3a is an evolutionary conserved molecule; it is present in such phylogenetically distant species as Drosophila and humans. Furthermore, the unique behavior of CC-3 on sections of normal, embryonic, and regenerative tissue and in cell culture immunostaining make it a reliable tool to identify mitotic foci.

Animals↗
PubMed1986

Human inhibin genes. Genomic characterisation and sequencing.

Inhibin is a gonadal hormone involved in the non-steroidal regulation of follicle stimulating hormone (FSH) secretion. Using the cDNAs coding for bovine inhibin A and B subunits we have identified inhibin genes within the human genome using Southern blot hybridisation techniques. The genes are likely to be present as single copies. Cloning and sequencing inhibin genes obtained from lambda libraries of human genomic DNA provide structural and sequence data on the human A and B genes. Comparison of the known inhibin gene sequences showed, in particular, that the B subunits have identical sequences in man, pigs and cattle thus demonstrating a remarkable evolutionary conservation in these genes.

Animals↗
PubMed1991

Histone H4 acetylation in Drosophila. Frequency of acetylation at different sites defined by immunolabelling with site-specific antibodies.

Electrophoresis, Western blotting and immunostaining with antibodies specific for histone H4 acetylated at lysines 5, 8, 12, or 16, were used to define patterns of H4 acetylation in cell lines from humans (HL60) and the fruit fly Drosophila (S2, Kc). In human cells, the mono-acetylated isoform H4Ac1 is acetylated predominantly at just one of the four possible lysine residues, lysine 16. This is the first step in the progressive acetylation of H4. In contrast, in Drosophila, H4Ac1 is acetylated at lysines 5, 8, or 12 with approximately equal frequency. Fundamental differences appear to exist in control of H4 acetylation in different species, despite the evolutionary conservation of acetylation sites.

Acetylation↗
PubMed1991

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis.

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.

Animals↗
PubMed1995

Homologues of the engrailed gene from five molluscan classes.

We used the polymerase chain reaction (PCR) to amplify, clone, and sequence 10 engrailed homeodomains from 8 species in the five major molluscan classes, including the serially organized chiton (Polyplacophora) lineage. The Drosophila melanogaster gene engrailed (en) is one of several genes involved in embryonic segment polarity determination. Studies of engrailed sequence and expression in molluscs are of interest due to questions regarding the evolution and homology of segmentation in these taxa. Nucleotide and deduced amino acid sequence comparisons reflect evolutionary conservation within helices of the en homeodomain and ancient divergences in the region 3' to the homeodomain.

Animals↗
PubMed1995

From entangled membranes to eclectic morphologies: cubic membranes as subcellular space organizers.

The identification of evolutionary conserved membrane morphologies whose architecture is governed by cubic symmetry--cubic membranes--adds a new dimension to cell membrane functions and, perspicuously, to their role in subcellular space organization. Through analysis of electron micrographs, three families of cubic membranes have been unequivocally identified in which one or more (parallel) membranes, described by periodic cubic surfaces, partition space into two or more independent, albeit convoluted, subspaces of membrane potential determined dimensions. The choice of a particular cubic symmetry is suggested to be due to its activity. Here the architecture and function of multiple (> or = 3) subspace organization in classical membrane bound organelles is addressed. As it can be precisely determined with cubic membranes suggests that they can be employed as a reference morphology.

Animals↗
PubMed1996

Towards prediction of cognate complexes between the WW domain and proline-rich ligands.

The WW domain is a structured protein module found in a wide range of regulatory, cytoskeletal, and signaling molecules. Its ligands contain proline-rich sequences, some of which show a core consensus of XPPXY that is critical for binding. In order to gain a better understanding of the molecular and biological functions of WW domains, we decided to predict their cognate ligands by searching databases for proteins containing the XPPXY consensus. Using several axioms that take into account evolutionary conservation and functional similarity, we have identified four groups of proteins representing candidate ligands that signal through known or unknown WW domains. These include viral Gag proteins, sodium channels, interleukin receptors, and a subgroup of serine/threonine kinases. In addition, we proposed that dystrophin and beta-dystroglycan bind through the WW-XPPXY link and that interference with this interaction could result in muscular dystrophy. Our study provides guidelines for experiments to probe the molecular and biological functions of the WW domain-ligand connection. Should these predictions be proven empirically, the results may have important ramifications for basic research and medicine.

Amino Acid Sequence↗
PubMed1986

Thyroid hormone binding to isolated trout (Salmo gairdneri) liver nuclei in vitro: binding affinity, capacity, and chemical specificity.

Isolated nuclei from trout hepatocytes demonstrated in vitro high-affinity and low-capacity binding for 3,5,3'-tri-iodo-L-thyronine (T3). Binding was reversible; the dissociation rate was 5.7 X 10(-3) min-1 at 15 degrees C. Linear Scatchard plots suggested a single class of noncooperative sites (Kd = 1.4 +/- 0.10 X 10(-10) M; maximum binding capacity (MBC) = 62 +/- 10 fmol/mg DNA). After correction for site degradation, site occupancy by endogenous T3, and the dissociation rate of endogenous bound T3, the MBC was 106 +/- 16 fmol/mg DNA. The T3 affinity exceeded slightly that of the hepatocyte nucleus of the rat; the MBC was lower than for most other vertebrates. The relative binding affinities of thyroid hormone (TH) analogs for the T3 site were: TRIPROP greater than TRIAC greater than methyl-bridged T3 greater than TETRAPROP greater than T3 greater than TETRAC greater than TRIFORM greater than 3,5-dibromo, 3'-isopropyl thyronine greater than L-thyroxine (T4) greater than DL-T4 greater than 3'-isopropyl 3',5'-dimethyl thyronine greater than reverse T3 greater than 3,5-T2. MIT and DIT did not bind at all. This structure-affinity profile was similar but not identical to that of rat liver, indicating considerable but not complete evolutionary conservation of site structure. Parallel studies of T4 binding also indicate a single class of noncooperative sites (Kd = 7.2 +/- 2.4 X 10(-10) M). Both the MBC and the structure-affinity profile for T4 corresponded to those for T3. These observations, combined with the ability of excess T3 or T4 to completely displace both labeled T3 or T4, support a previous suggestion that in teleosts T3 and T4 bind to the same class of nuclear sites. These sites probably represent TH receptors.

Animals↗
PubMed1985

Structure and genomic organization of the rat aldolase B gene.

The structure of the chromosomal gene encoding rat aldolase isozyme B has been elucidated by sequence analysis of cloned genomic DNA. This gene comprises about 14 X 10(3) base-pairs of DNA, and is separated into nine exons by eight intervening sequences. A presumed transcription-initiation site was assigned by S1 nuclease protection mapping, and T-A-T-A and C-C-A-A-T boxes were found to be 25 and 126 base-pairs, respectively, upstream from this initiation site. There are three characteristic sequences of 100 to 200 base-pairs within the region of 870 base-pairs flanking the 5' side of the gene. These sequences are flanked on either side by direct repeats and terminate with an A-rich stretch of nucleotides. One of them has block homology with a region in an "ID sequence", which is reported to be an element for tissue-specific gene regulation and differentiation. The other two are analogous at the sequence organizational level with a sort of dispersed repeat, the "Alu family". These features suggest that these regions are involved in gene regulation and, also, imply evolutionary events such as duplication or insertion. Comparison of this gene sequence with the rabbit aldolase A complementary DNA sequence revealed some bias in the frequency of nucleotide replacement among the exons, suggesting selective evolutionary conservation of particular exons encoding functional domains. Comparison with the human aldolase B complementary DNA sequence revealed no such tendency; the homology between the two sequences was very high (about 89%), and nucleotide replacements were randomly distributed throughout the protein-coding region.

Animals↗
PubMed1988

Estrogen modulation of riboflavin carrier protein in the bonnet monkey (Macaca radiata).

Circulatory concentrations of riboflavin carrier protein (RCP) were quantitated in bonnet macaques by employing a heterologous radioimmunoassay involving 125I-labelled chicken RCP and its antiserum. The levels of monkey RCP in the serum seem to be governed by the estrogenic status of the animals. An increase in concentration of serum estradiol in the adult females during the menstrual cycle and early pregnancy could be correlated with enhanced serum RCP levels. Estadiol-17 beta administered to both immature female and male monkeys, specifically brought about elevated levels of RCP with a slower time course of response in males than in females. These results could be a reflection of a more rapid decline of both circulatory estrogen and RCP concentrations in male serum. Repeated administration of estradiol-17 beta to male animals led to prolonged elevated levels of RCP following estrogen administration. Thus, it would appear that the evolutionary conservation of RCPs from the aves to the primates encompasses not only their physicochemical similarities but also extends to the estrogenic modulation of their elaboration.

Animals↗
PubMed1994

Serine/threonine protein phosphatases in the control of cell function.

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Amino Acid Sequence↗