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C Ferraz

Publications and source records attributed to C Ferraz.

At least 37 records · Page 2Linked to original sources
PubMed1991

Distribution of gelsolin in the retina of the developing rabbit.

The distribution of gelsolin, a calcium-dependent actin-severing and capping protein, in the retina of the developing and adult rabbit was studied. Gelsolin immunoreactivity was found in the photoreceptors and ganglion cells, where it may have a role in neuronal morphogenesis. Only the inner segment of the photoreceptors retained a high gelsolin content in the adult retina, perhaps because the attached outer segment is continuously renewed throughout life. Gelsolin, which is a major component of the rabbit brain oligodendrocytes, was also found in the myelin of the medullary ray region of the rabbit retina. Müller cells in all regions of the rabbit retina also contain gelsolin from early in development to adulthood. Since one of the functions of these cells is to ensheath neuronal elements in the inner plexiform and optic fiber layers, we suggest that gelsolin may play the same role in Müller cells as it does in oligodendrocytes, i.e., sheath formation via its calcium-dependent action on the actin microfilament networks.

Animals↗
PubMed1991

Conformational stability of human skeletal tropomyosins modified by site-directed mutagenesis.

We have used human beta-tropomyosin produced in Escherichia coli and deletion mutants obtained by site-directed mutagenesis to analyse the conformational stability of this molecule under various experimental conditions. Protein engineering has allowed us to answer some questions raised by stability analysis of the wild-type tropomyosin. The complex pattern of denaturation is due neither to heterogeneity of the preparation nor to head-to-tail interactions. The N- and C-termini are not of importance for the thermal stability of the molecule. On the contrary, deletion of the 31 C-terminus amino acids leads to a dramatic decrease of the stability observed in guanidinium chloride. This lowering is interpreted as the participation of one more guanidinium chloride ions to the denaturation equilibrium. Analysis of the stability in presence of organic solvents reveals that acetonitrile and methanol induce opposite effects. Investigation of these effects by three methods (CD, fluorescence and electrophoresis that measure respectively the content in alpha-helix, the contact between the two strands and the strands exchange) leads to the conclusion that strand separation can precede the denaturation of the alpha-helix.

Circular Dichroism↗
PubMed1991

Profilin and profilin mRNA in the cerebellum of the developing rat.

Profilin and profilin mRNA concentrations were determined in the cerebellum of the developing rat. Profilin represented nearly 0.1% of total Triton-soluble protein both at birth and at the end of the developmental period. During brain maturation, the profilin concentration was regulated at the transcriptional level. A transient increase of profilin mRNA and profilin was observed at the end of the first postnatal week, a period of intense cell proliferation, neuritic outgrowth and synaptogenesis. We propose that profilin may act by forming, during the critical period of cerebellar development, a reserve pool of monomeric actin that can easily be mobilized in cell regions such as growth cones or synaptic junctions where filamentous actin is highly concentrated.

Actins↗
PubMed1990

Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9).

The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.

Actins↗
PubMed1990

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding.

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.

Amino Acid Sequence↗
PubMed1990

Effect of thyroid deficiency on actin mRNA content in the developing rat cerebellum.

The actin mRNA content of the cerebellum was determined in normal and hypothyroid developing rats using RNA dot hybridization with a beta-actin cDNA probe. The decline in actin mRNA content occurring during the second postnatal week in normal development was delayed by about 1 week in hypothyroid rats. Since this effect coincides exactly with the delay in actin filament formation recently reported in thyroid-deficient rats, it strengthens the hypothesis of an inverse relationship in the developing brain between the polymerization state of actin and the production of actin mRNA.

Actins↗
PubMed1990

Quantitative analysis of the selective pressure exerted on homologous proteins.

Evolution analysis is used to locate the regions of a protein that are important for its function or structure. The rate of evolution is generally constant for a given family of homologous sequences. From the starting point of this observation, an algorithm is proposed to establish quantitatively the sequence zones where selective pressure is maximal. A program that computes this pressure has been written in PASCAL. Analysis of results on some sequences validate this theoretical approach, and this knowledge can be used as a starting-point for carrying out site-directed mutagenesis.

Algorithms↗
PubMed1990

Expression of tropomyosin genes during the development of the rat cerebellum.

The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human-specific alpha-, beta-, gamma-, and delta-tropomyosin cDNA probes and rat-specific alpha-, beta-, and delta-tropomyosin oligonucleotide probes. The beta- and gamma-genes do not seem to be expressed in the rat brain. The delta-tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4-kb mRNA. A 3-kb mRNA is expressed by the alpha-tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.

Animals↗
PubMed1989

Purification of synthetic oligonucleotides on a weak ion-exchange column.

A weak ion-exchange column (PVDI 4000-5) was used to purify oligonucleotides of relatively large size. The purification of polynucleotides was very rapid, they were separated according to size and retained all their properties relevant to genetic engineering experiments.

Chromatography, Ion Exchange↗
PubMed1989

Cloning and deletion mutagenesis using direct protein-protein interaction on an expression vector. Identification of the calmodulin binding domain of alpha-fodrin.

We have screened a lambda gt11 library, constructed with mouse macrophage cDNA, in order to isolate clones that code for calmodulin binding proteins. We have developed a new approach for this purpose using radioactive calmodulin (produced by genetic engineering) to detect fusion proteins that interact with this protein with high affinity. A cDNA clone that codes for mouse macrophage fodrin was isolated, sequenced and identified. By deleting part of the sequence the calmodulin binding domain was located on the fodrin sequence. The site is situated on repeat 11 of fodrin and probably on the extra arm of this repeat. The method we developed is widely applicable to site-directed mutagenesis of interacting proteins.

Amino Acid Sequence↗
PubMed1989

Structural analysis of human skeletal beta-tropomyosin produced in E. coli.

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

Amino Acid Sequence↗
PubMed1988

Analysis of double-stranded poly(A).poly(U) molecules by reversed-phase high-performance liquid chromatography.

The behaviour of different batches of synthetic Poly(A).Poly(U) in reversed-phase high-performance liquid chromatography (HPLC) was studied. They consist of large molecules mainly in the form of a double strand. Differences in the elution patterns were correlated with properties detected by conventional methods such as electrophoresis, centrifugation, fusion analysis or enzymatic digestions. Under the present conditions, contamination by products and precursors used during synthesis was detectable, but was absent in most of the preparations. The differences in elution patterns between batches appear to be correlated with the size of the molecules synthesized. The chromatograms suggested that Poly(A).Poly(U) molecules contain single-strand portions at least transiently. The presence of such portions was confirmed by enzymatic digestion with S1 nuclease. The rapidity, reproducibility and ease of reversed-phase HPLC qualify this technique as a tool for routine analysis.

Chromatography, High Pressure Liquid↗
PubMed1988

Gelsolin immunoreactivity and development of the tectorial membrane in the cochlea of normal and hypothyroid rats.

Gelsolin was localized by immunocytochemistry in the developing cochlea of the rat. In normal animals, the protein appeared at 18 th day in utero in cells of the Kölliker's organ, which are involved in the secretion of the tectorial membrane. The Kölliker's organ cells were not immunoreactive after the first postnatal week, which is when they cease their secretory activity. Gelsolin immunoreactivity was similar in thyroid-deficient rats until the second postnatal week but, at this age, Kölliker's organ did not transform and its gelsolin immunoreactivity persisted, together with its secretory activity. As a result, the tectorial membrane was greatly distorted and out of contact with the hair cells, which dramatically impaired the mechanical properties of the organ of Corti. The developing cochlea thus provides an example of the involvement of gelsolin in a secretory process that is of importance in the development of hearing.

Animals↗
PubMed1988

Evolution of tropomyosin functional domains: differential splicing and genomic constraints.

We have cloned and determined the nucleotide sequence of a complementary DNA (cDNA) encoded by a newly isolated human tropomyosin gene and expressed in liver. Using the least-square method of Fitch and Margoliash, we investigated the nucleotide divergences of this sequence and those published in the literature, which allowed us to clarify the classification and evolution of the tropomyosin genes expressed in vertebrates. Tropomyosin undergoes alternative splicing on three of its nine exons. Analysis of the exons not involved in differential splicing showed that the four human tropomyosin genes resulted from a duplication that probably occurred early, at the time of the amphibian radiation. The study of the sequences obtained from rat and chicken allowed a classification of these genes as one of the types identified for humans. The divergence of exons 6 and 9 indicates that functional pressure was exerted on these sequences, probably by an interaction with proteins in skeletal muscle and perhaps also in smooth muscle; such a constraint was not detected in the sequences obtained from nonmuscle cells. These results have led us to postulate the existence of a protein in smooth muscle that may be the counterpart of skeletal muscle troponin. We show that different kinds of functional pressure were exerted on a single gene, resulting in different evolutionary rates and different convergences in some regions of the same molecule. Codon usage analysis indicates that there is no strict relationship between tissue types (and hence the tRNA precursor pool) and codon usage. G + C content is characteristic of a gene and does not change significantly during evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Under our experimental conditions, the retention time depends not on the chain length but rather on the base composition and the secondary structure of the molecule. Because of the torsional strain caused by the supercoiling of the plasmid, more of its bases are accessible for interaction with the hydrophobic stationary phase. This increases the retention time of the supercoiled DNA compared to the relaxed or linear DNA. We have exploited these properties to analyze the quality of plasmid preparations. The method is more sensitive to contaminants than common electrophoretic techniques. Furthermore, we describe a convenient and rapid procedure for purifying plasmid DNA. The highly pure plasmid is biologically more active for most of the enzymatic reactions commonly used in genetic engineering.

Base Composition↗
PubMed1986

Immunocytochemical localisation of gelsolin in oligodendroglia of the developing rabbit central nervous system.

Antibodies raised against gelsolin of rabbit lung macrophages were used in an immunocytochemical study during development of the rabbit central nervous system. With the exception of a transient staining of the cerebellar Purkinje cells during the first postnatal week, gelsolin was found a new marker for oligodendrocytes, especially in developing animals. In the macrophages, gelsolin is involved in the control of locomotion, secretion and endocytosis. In the oligodendrocytes, which produce the myelin sheaths in the central nervous system, gelsolin could therefore be involved in the control of the complex motile events leading to myelin wrapping.

Animals↗