Biomedical subjects

M J Howard

Publications and source records attributed to M J Howard.

At least 19 recordsLinked to original sources

PubMed2001

The ligand-binding loops in the tunicate C-type lectin TC14 are rigid.

C-type lectin-like domains are very common components of extracellular proteins in animals. They bind to a variety of ligands, including carbohydrates, proteins, ice, and CaCO3 crystals. Their structure is characterized by long surface loops in the area of the protein usually involved in ligand binding. The C-type lectin TC14 from Polyandrocarpa misakiensis specifically binds to D-galactose by coordination of the sugar to a bound calcium atom. We have studied the dynamic properties of TC14 by measuring 15N longitudinal and transverse relaxation rates as well as [1H-15N] heteronuclear NOEs. Relaxation rates and heteronuclear NOE data for holo-TC14 show minimal variations, indicating that there is no substantial difference in rigidity between the elements of regular secondary structure and the extended surface loops. Anisotropic tumbling of the elongated TC14 dimer can account for the main fluctuations in relaxation rates. Loss of the bound calcium does not significantly alter the internal dynamics, suggesting that the stability of the loop region is intrinsic and not dependent on the coordination of the calcium ion. Chemical shift differences between the holo and apo form show that main structural changes occur in the calcium-binding site, but smaller structural changes are propagated throughout the molecule without affecting the overall fold. The disappearance of two resonances for residues following the conserved cis-proline 87 (which is located in the calcium-binding site) in the apo form indicates conformational change on an NMR time scale between the cis and trans configurations of this peptide bond in the absence of calcium. Possible implications of these findings for the ligand binding in C-type lectin-like domains are discussed.

Animals↗

PubMed2001

The UBX domain: a widespread ubiquitin-like module.

The UBX domain is an 80 amino acid residue module that is present typically at the carboxyl terminus of a variety of eukaryotic proteins. In an effort to elucidate the function of UBX domains, we solved the three-dimensional structure of the UBX domain of human Fas-associated factor-1 (FAF1) by NMR spectroscopy. The structure has a beta-Grasp fold characterised by a beta-beta-alpha-beta-beta-alpha-beta secondary-structure organisation. The five beta strands are arranged into a mixed sheet in the order 21534. The longer first helix packs across the first three strands of the sheet, and a second shorter 3(10) helix is located in an extended loop connecting strands 4 and 5. In the absence of significant sequence similarity, the UBX domain can be superimposed with ubiquitin with an r.m.s.d. of 1.9 A, suggesting that the two structures share the same superfold, and an evolutionary relationship. However, the absence of a carboxyl-terminal extension containing a double glycine motif and of suitably positioned lysine side-chains makes it highly unlikely that UBX domains are either conjugated to other proteins or part of mixed UBX-ubiquitin chains. Database searches revealed that most UBX domain-containing proteins belong to one of four evolutionarily conserved families represented by the human FAF1, p47, Y33K, and Rep8 proteins. A role of the UBX domain in ubiquitin-related processes is suggested.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Two signal transduction pathways involved in the catecholaminergic differentiation of avian neural crest-derived cells in vitro.

Molecules derived from the neural tube and found in chick embryo extract (CEE) and bone morphogenetic proteins (BMP) support the differentiation of neural crest-derived catecholaminergic (CA) neurons. We now report that intracellular signaling resulting in the activation of Map kinase (MapK) or translocation of Smad1 mediate the differentiation of CA neurons in response to CEE or BMP 4, respectively. The differentiation of CA neurons was significantly reduced by inhibiting MapK using PD98059 or by pan-specific blockade of tyrosine kinases using Herbimycin A. In the presence of BMP 4 and inhibitors of MapK signaling, differentiation of CA neurons was only moderately reduced. Independent of MapK, BMP 4 induced translocation of Smad1 from the cytosol to the nucleus and induced transcription of dHAND, a DNA binding protein required for the differentiation of CA neurons. The data suggest that CEE-derived factors and BMP4 support the differentiation of CA neurons via independent signaling pathways.

Animals↗

PubMed2000

Elucidating the mechanism of familial amyloidosis- Finnish type: NMR studies of human gelsolin domain 2.

Familial amyloidosis-Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between beta-strand 4 and alpha-helix 1 and within the loop region following beta-strand 5. Chemical shift differences are noted along the face of alpha-helix 1 that packs onto the beta-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. [(1)H-(15)N] nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

Amyloid Neuropathies↗

PubMed2000

Biophysical characterization of elongin C from Saccharomyces cerevisiae.

Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.

Amino Acid Sequence↗

PubMed2000

Trithiacyclononane as a ligand for potential technetium and rhenium radiopharmaceuticals: synthesis of [M(9S3)(SC2H4SC2H4S)][BF4] (M = 99Tc, Re, 188Re) via C-S bond cleavage.

Chemical or electrochemical reduction of the 1,4,7-trithiacyclononane (9S3) complexes [MII(9S3)2][BF4]2 (M = Re (3a) or Tc (3b)) results in instantaneous C-S bond cleavage to yield ethene and the stable MIII thiolate complexes [MIII(9S3)L][BF4] (M = Re (4a) or Tc (4b), L = SCH2CH2SCH2CH2S). Compounds 4 have been characterized by 1H NMR spectroscopy, and the pseudo-octahedral geometry of 4b has been confirmed by X-ray crystallography. Upon electrochemical reduction 4a loses ethene, while 4b can be reversibly reduced to [TcII(9S3)L], which is then further reduced to Tc(I) with loss of ethene. Successive ethene loss is observed in the mass spectra of compounds 3 and 4. The radiosynthesis of 4a with 188Re can be comfortably completed within 10 min starting with 188ReO4- from a 188W/188Re generator, with a radiochemical yield in excess of 90%, and thus represents a practical approach to the preparation of stable 188Re (and 99mTc) thioether complex derivatives/conjugates for clinical use. Crystal data: 4b, C10H20S6Tc, orthorhombic Pbca, a = 12.233(2) A, b = 14.341(2) A, c = 20.726(3) A, Z = 8.

Alkanes↗

PubMed2000

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.

Acetyl-CoA Carboxylase↗

PubMed2000

Restricted motion of the lipoyl-lysine swinging arm in the pyruvate dehydrogenase complex of Escherichia coli.

The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase (PDH) complex of Escherichia coli house the lipoyl-lysine side chain essential for active-site coupling and substrate channelling within the complex. The structure of the unlipoylated form of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybrid domain determined previously [Green et al. (1995) J. Mol. Biol. 248, 328-343]. The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the sheets; the N- and C-termini lie close together at the opposite end of the molecule in the other beta-sheet. Measurement of (15)N NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the residues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue. This implies that the lipoyl-lysine side chain may not sample the full range of conformational space once thought. Moreover, reductive acetylation of the lipoylated domain (E2plip(holo) --> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonances were observed for several residues. This implies a change in conformation and the existence of multiple conformations of the domain on reductive acetylation, which may be important in stabilizing this catalytic intermediate.

Acylation↗

PubMed2000

Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Enhanced remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cells suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. In this paper we show that oligodendrocyte cultures can be reliably produced from retinoic acid-induced ES cells and that these oligodendrocytes can myelinate axons in vitro. Methods were further developed for generating highly enriched cultures of oligodendrocytes through an additional culturing step, producing an intermediate "oligosphere" stage. To test whether ES cells can survive, migrate, and differentiate into mature myelin-producing cells in areas of demyelination in the adult CNS, ES cells were transplanted into the dorsal columns of adult rat spinal cord 3 days after chemical demyelination. In the demyelination site, large numbers of ES cells survived and differentiated primarily into mature oligodendrocytes that were capable of myelinating axons. Furthermore, when oligosphere cells were transplanted into the spinal cords of myelin-deficient shiverer (shi/shi) mutant mice, the ES cell-derived oligodendrocytes migrated into the host tissue, produced myelin and myelinated host axons. These studies demonstrate the ability of ES cell-derived oligodendrocytes to myelinate axons in culture and to replace lost myelin in the injured adult CNS. Transplantation of ES cells may be a practical approach to treatment of primary and secondary demyelinating diseases in the adult CNS.

Animals↗

PubMed2000

Protein-protein interaction revealed by NMR T(2) relaxation experiments: the lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

T(2) relaxation experiments in combination with chemical shift and site-directed mutagenesis data were used to identify sites involved in weak but specific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase component, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first committed and irreversible catalytic step. The accompanying reductive acetylation of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between E1 and the lipoyl domain of the E2 polypeptide chain. The interactions between the free lipoyl domain (9 kDa) and free E1alpha (41 kDa), E1beta (35 kDa) and intact E1alpha(2)beta(2) (152 kDa) components, all the products of genes or sub-genes over-expressed in Escherichia coli, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uniformly (15)N-labeled lipoyl domain and unlabeled E1 components. Major contact points on the lipoyl domain were identified from changes in the backbone (15)N spin-spin relaxation time in the presence and absence of E1(alpha(2)beta(2)) or its individual E1alpha or E1beta components. Although the E1alpha subunit houses the sequence motif associated with the essential cofactor, thiamin diphosphate, recognition of the lipoyl domain was distributed over sites in both E1alpha and E1beta. A single point mutation (N40A) on the lipoyl domain significantly reduces its ability to be reductively acetylated by the cognate E1. None the less, the N40A mutant domain appears to interact with E1 similarly to the wild-type domain. This suggests that the lipoyl group of the N40A lipoyl domain is not being presented to E1 in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain structure, especially in the lipoyl-lysine beta-turn region, as indicated by chemical shift data. Interaction with E1 and subsequent reductive acetylation are not necessarily coupled.

Amino Acid Sequence↗

PubMed2000

The transcription factor dHAND is a downstream effector of BMPs in sympathetic neuron specification.

The dHAND basic helix-loop-helix transcription factor is expressed in neurons of sympathetic ganglia and has previously been shown to induce the differentiation of catecholaminergic neurons in avian neural crest cultures. We now demonstrate that dHAND expression is sufficient to elicit the generation of ectopic sympathetic neurons in vivo. The expression of the dHAND gene is controlled by bone morphogenetic proteins (BMPs), as suggested by BMP4 overexpression in vivo and in vitro, and by noggin-mediated inhibition of BMP function in vivo. The timing of dHAND expression in sympathetic ganglion primordia, together with the induction of dHAND expression in response to Phox2b implicate a role for dHAND as transcriptional regulator downstream of Phox2b in BMP-induced sympathetic neuron differentiation.

Animals↗

PubMed2000

A light microscopy study of the migration of Naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice.

The migratory pathway of Naegleria fowleri from the nasal submucosa to the central nervous system (CNS) during the early stage of primary amebic meningoencephalitis (PAM) was investigated in mice. Twenty-one-day-old CD-1 mice were inoculated by intranasal instillation of 1 x 10(6) amebas. Animals were divided into 3 groups of 5 and, after being anesthetized, were killed at intervals of 24, 32, and 48 hr postinoculation by transcardial perfusion with formaldehyde, acetic acid, and methanol. The heads were decalcified, divided in the midsagittal plane, and the area of the cribriform plate removed and embedded in paraffin. Serial sections were cut at 8 microm and stained with a combination of celestin blue, Harris' hematoxylin, and acid fuchsin for light microscopy. Focal inflammation and amebas were observed in the submucosal nerve plexus, olfactory nerves penetrating the cribriform plate, and the olfactory bulb of the brain as early as 24 hr postinoculation. The time periods selected assured that the disease process would not obliterate soft tissue structures. Earlier studies used moribund mice in which the inflammation and the number of amebas were overwhelming. The present study provides convincing evidence that amebas gain initial access to the CNS through olfactory nerves within the cribriform plate during the early stages of PAM.

Amebiasis↗

PubMed1999

The effect of matrix metalloproteinase complex formation on the conformational mobility of tissue inhibitor of metalloproteinases-2 (TIMP-2).

The backbone mobility of the N-terminal domain of tissue inhibitor of metalloproteinases-2 (N-TIMP-2) was determined both for the free protein and when bound to the catalytic domain of matrix metalloproteinase-3 (N-MMP-3). Regions of the protein with internal motion were identified by comparison of the T(1) and T(2) relaxation times and (1)H-(15)N nuclear Overhauser effect values for the backbone amide (15)N signals for each residue in the sequence. This analysis revealed rapid internal motion on the picosecond to nanosecond time scale for several regions of free N-TIMP-2, including the extended beta-hairpin between beta-strands A and B, which forms part of the MMP binding site. Evidence of relatively slow motion indicative of exchange between two or more local conformations on a microsecond to millisecond time scale was also found in the free protein, including two other regions of the MMP binding site (the CD and EF loops). On formation of a tight N-TIMP-2. N-MMP-3 complex, the rapid internal motion of the AB beta-hairpin was largely abolished, a change consistent with tight binding of this region to the MMP-3 catalytic domain. The extended AB beta-hairpin is not a feature of all members of the TIMP family; therefore, the binding of this highly mobile region to a site distant from the catalytic cleft of the MMPs suggests a key role in TIMP-2 binding specificity.

Amino Acid Sequence↗

PubMed1999

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy.

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.

Acetyl-CoA Carboxylase↗

PubMed1999

Resealing of transected myelinated mammalian axons in vivo: evidence for involvement of calpain.

The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5 microm outer diameter) resealed faster than larger axons. Resealing was Ca2+ dependent, requiring micromolar levels of extracellular [Ca2+] to proceed, and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in saline containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BAPTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+ -activated proteases. Resealing in 2 mM Ca2+ was not inhibited by an inhibitor of phospholipase A2. Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.

Animals↗

PubMed1999

SINNMR studies of acoustically induced rotation of suspended particles.

Experiments and data analyses are reported on the 20 kHz acoustic manipulation of samples of trisodium phosphate dodecahydrate, of varying particle size, suspended in suitable support media. Data are obtained to expand the understanding and optimisation of the SINNMR (sonically induced narrowing of the nuclear magnetic resonance spectra of solids) technique, and use this to determine acoustically induced particle rotational correlation times. It is concluded that the average particle rotational correlation time decreases with increasing particle size and decreasing viscosity/density of the support media. Acoustic cavitation and accompanying interparticle collisions are shown to be important in the observation of SINNMR spectra.

Journal Article↗

PubMed1999

Hantavirus pulmonary syndrome in pregnancy.

This comprehensive case review of hantavirus pulmonary syndrome (HPS) during pregnancy in 5 women characterizes the effect of Sin Nombre virus infection on maternal and fetal outcomes. Histopathologic, serological, and clinical information were evaluated for evidence of vertical transmission. Maternal ages ranged from 20 to 34 years and gestational ages from 13 to 29 weeks. Symptoms, physical findings, and laboratory values other than those related to pregnancy were not noticeably different from those of nonpregnant patients with HPS, although fevers were somewhat lower. One maternal death and 2 fetal losses occurred. Gross, microscopic, and immunohistochemical examination for hantavirus antigen were done on 2 fetal autopsies and 3 placentas showing no evidence of transplacental hantavirus transmission. There was no serological evidence of conversion in the 3 surviving children. Maternal and fetal outcomes of HPS appear similar to those of nonpregnant HPS patients and of pregnant patients with other causes of acute respiratory distress syndrome. No evidence of vertical transmission of Sin Nombre virus was found.

Adult↗

PubMed1998

Insulin stimulates pp120 endocytosis in cells co-expressing insulin receptors.

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.

3T3 Cells↗