PubMed Health⌕ Search

Biomedical subjects

J M Thorn

Publications and source records attributed to J M Thorn.

17 recordsLinked to original sources
PubMed2001

The NIEHS Xenopus maternal EST project: interim analysis of the first 13,879 ESTs from unfertilized eggs.

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.

Alleles↗
PubMed2000

Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways.

Intersectin is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. In general, EH domain-containing proteins play an integral role in clathrin-mediated endocytosis. Indeed, intersectin functions in the intermediate stages of clathrin-coated vesicle assembly. However, recent evidence suggests that components of the endocytic machinery also regulate mitogenic signaling pathways. In this report, we provide several lines of evidence that intersectin has the capacity to activate mitogenic signaling pathways. First, intersectin overexpression activated the Elk-1 transcription factor in an MAPK-independent manner. This ability resides within the EH domains, as expression of the tandem EH domains was sufficient to activate Elk-1. Second, intersectin cooperated with epidermal growth factor to potentiate Elk-1 activation; however, a similar level of Elk-1 activation was obtained by expression of the tandem EH domains suggesting that the coiled-coil region and SH3 domains act to regulate the EH domains. Third, intersectin expression was sufficient to induce oncogenic transformation of rodent fibroblasts. And finally, intersectin cooperated with progesterone to accelerate maturation of Xenopus laevis oocytes. Together, these data suggest that intersectin links endocytosis with regulation of pathways important for cell growth and differentiation.

3T3 Cells↗
PubMed2000

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA.

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.

3' Untranslated Regions↗
PubMed1999

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression.

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

Amino Acid Sequence↗
PubMed1999

Identification and characterisation of Xenopus moesin, a Src substrate in Xenopus laevis oocytes.

The cortical actin cytoskeleton, consisting of actin filaments and actin binding proteins, immediately underlies the inner surface of the plasma membrane and is important both structurally and in relaying signals from the surface to the interior of the cell. Signal transduction processes, initiated in the cortex, modulate numerous cellular changes ranging from modifications of the local cytoskeleton structure, the position in the cell cycle, to cell behaviour. To examine the molecular mechanisms and events associated with cortical changes. We have investigated targets of the protein tyrosine kinase, Src, which is associated with the cortical cytoskeleton, in Xenopus laevis oocytes. When a mRNA encoding an activated form of Src tyrosine kinase (d-Src) is injected into oocytes several changes are observed: proteins are phosphorylated, the rate at which progesterone matures an oocyte to an egg is accelerated, and the cortex at the site of injection appears to contract. Previous studies have implicated actin filaments in the Src-stimulated cortical rearrangements. In this study we identify two actin binding proteins-cortactin and moesin--as Src substrates in Xenopus oocytes that are Src substrates. We cloned and characterised the cDNA encoding one of those, Xenopus moesin, a member of the ezrin/radixin/moesin (ERM) family of actin binding proteins. In addition, we have determined that moesin is recruited to the cortex at the site of Src mRNA injection.

Actins↗
PubMed1998

Reliability of delta-crystallin as a marker for studies of chick lens induction.

Induction of a lens by the optic vesicle of the brain was the first demonstration of how tissue interactions could influence cell fate during development. However, recent work with amphibians has shown that the optic vesicle is not the primary inducer of lens formation. Rather, an earlier interaction between anterior neural plate and presumptive lens ectoderm appears to direct lens formation. One problem with many early experiments was the absence of an unambiguous assay for lens formation. Before being able to test whether the revised model of lens induction applies to chicken embryos, we examined the suitability of using delta-crystallin as a marker of lens formation. Although delta-crystallin is the major protein synthesized in the chick lens, one or both of the two delta-crystallin genes found in chickens is transcribed in many non-lens tissues as well. In studies of lens formation where appearance of the delta-crystallin protein is used as a positive assay, synthesis of delta-crystallin outside of the lens could make experiments difficult to interpret. Therefore, polyacrylamide gel electrophoresis, immunoblotting, and immunofluorescence were used to determine whether the delta-crystallin messenger RNA detected in non-lens tissues is translated into protein, as it is in the lens. On Coomassie-blue-stained gels of several tissues from stage-22 embryos, a prominent protein was observed that co-migrated with delta-crystallin. However, on immunoblots, none of the nonlens tissues tested contained detectable levels of delta-crystallin at this stage. By imunofluorescence, delta-crystallin was observed in Rathke's pouch and in a large area of oral ectoderm near Rathke's pouch, yet none of the cells in these non-lens tissues showed the typical elongated morphology of lens fiber cells. When presumptive lens ectoderm or other regions of ectoderm from stage-10 embryos were cultured and tested for lens differentiation, both cell elongation and delta-crystallin synthesis were observed, or neither were observed. The results suggest that delta-crystallin synthesis and cell elongation together serve as useful criteria for assessing a positive lens response.

Animals↗
PubMed1996

Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases.

Quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases. The crystal structures of Escherichia coli quinone oxidoreductase (QOR) and Thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase. A structurally based comparison of these three enzymes confirms that they possess extensive overall structural homology despite low sequence identity. The most significant difference is the absence of the catalytic and structural zinc ions in QOR. A multiple structure-based sequence alignment has been constructed for the three enzymes and extended to include zeta-crystallin, an eye lens structural protein with quinone oxidoreductase activity and high sequence identity to E. coli quinone oxidoreductase. Residues which are important for catalysis have been altered and the functions and activities of the enzymes have diverged, illustrating a classic example of divergent evolution among a superfamily of enzymes.

Alcohol Dehydrogenase↗
PubMed1995

Crystal structure of Escherichia coli QOR quinone oxidoreductase complexed with NADPH.

The crystal structure of the homodimer of quinone oxidoreductase from Escherichia coli has been determined using the multiple isomorphous replacement method at 2.2 A resolution and refined to an R-factor of 14.1% The crystallographic asymmetric unit contains one functional dimer with the two subunits being related by a non-crystallographic 2-fold symmetry axis. The model consists of two polypeptide chains (residues 2 through 327), one NADPH molecule and one sulphate anion per subunit, and 432 water molecules. Each subunit consists of two domains: a catalytic domain and a nucleotide-binding domain with the NADPH co-factor bound in the cleft between domains. Quinone oxidoreductase has an unusual nucleotide-binding fingerprint motif consisting of the sequence AXXGXXG. The overall structure of quinone oxidoreductase shows strong structural homology to that of horse liver alcohol dehydrogenase.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries.

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.

3T3 Cells↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies on a soluble Escherichia coli quinone oxidoreductase.

Single crystals suitable for X-ray diffraction studies have been obtained from a soluble Escherichia coli NAD(P)H-dependent quinone oxidoreductase (QOR) of molecular mass 35 kDa. Crystals were grown by the vapour diffusion method in the presence of NADPH and diffract strongly to beyond 2.0 A. The space group was determined to be P2(1)2(1)2(1) with unit cell dimensions of a = 107.44 A, b = 104.06 A, c = 77.45 A. There is one dimer in the asymmetric unit.

Crystallization↗
PubMed1994

Crystallization and low temperature diffraction studies of the DNA binding domain of the single-stranded DNA binding protein from Escherichia coli.

The DNA binding domain of the single-stranded DNA binding protein from Escherichia coli has been overproduced, purified and crystallized in a form suitable for X-ray diffraction studies. Crystals were produced by dialysis against low ionic strength buffer at high pH. The crystals belong to space group I222 or I2(1)2(1)2(1) with a = 82.47 A, b = 65.27 A and c = 46.50 A. Data were collected at several temperatures and a significant improvement in data quality was observed with a decrease in temperature. On occasion, with the decrease in temperature, a transformation to a primitive space group was observed and temperature appears to play a key role in this transformation. A complete native data set has been collected to 2.57 A at -15 degrees C.

Bacterial Proteins↗
PubMed1992

Purification of maize pollen exines and analysis of associated proteins.

Zea mays (maize) pollen exines have been purified with the use of differential centrifugation and sucrose gradients, followed by mild detergent and high salt treatment. The final exine fraction is highly purified from other organelles and subcellular structures as assayed by transmission electron microscopy. Using mature maize pollen as the starting material, 0.2 to 0.3% of the total pollen protein remained associated with the exine fraction throughout the purification. Seven abundant sodium dodecyl sulfate-extractable proteins are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the final fraction. Amino acid analysis reveals that one of the proteins contains a substantial amount of hydroxyproline, a characteristic of some primary cell wall proteins. The amino acid composition of the 25-kD protein strongly implies that it is an arabinogalactan protein. When exines are purified from earlier pollen developmental stages, a subset of the proteins found in the mature pollen exine is seen.

Journal Article↗
PubMed1983

Evaluation of the trace metal supplements for a synthetic low lactose diet.

A trace element supplement used with a synthetic low lactose milk (Galactomins 17 and 18) has been evaluated by means of metabolic balance studies in 4 infants with dissacharide intolerances. The supplement was considered satisfactory for iron and manganese but increases in its zinc and copper content are probably necessary to ensure adequate retentions of these metals.

Body Weight↗
PubMed1981

The therapeutic effect of amphotericin in acrodermatitis enteropathica: hypothesis and implications.

A nine year-old girl with acrodermatitis enteropathica developed typical clinical and biochemical features of zinc deficiency on two occasions while on an oral zinc supplement. On both occasions, these features responded immediately when she was treated with amphotericin B lozenges. Studies in vitro showed that amphotericin increases the permeability to zinc of pure lipid membranes containing cholesterol. We suggest that the antibiotic enhanced zinc absorption from the oral supplement thereby effecting resolution of the patient's zinc deficiency.

Acrodermatitis↗
PubMed1978

Mineral and trace metal supplement for use with synthetic diets based on comminuted chicken.

Earlier studies (Alexander et al., 1974; Lawson et al., 1977) suggested a suitable composition for a mineral and trace metal supplement for use with synthetic diets containing some natural food. Such a mixture has been evaluated in patients receiving a diet based on comminuted chicken and has been shown to be adequate. This conclusion was based on balance experiments measuring Zn, Cu, Mn, Fe, Ca, Mg, N, and P.

Animals↗