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PubMed2006

Expression, limited proteolysis and preliminary crystallographic analysis of IpaD, a component of the Shigella flexneri type III secretion system.

IpaD, the putative needle-tip protein of the Shigella flexneri type III secretion system, has been overexpressed and purified. Crystals were grown of the native protein in space group P2(1)2(1)2(1), with unit-cell parameters a = 55.9, b = 100.7, c = 112.0 A, and data were collected to 2.9 A resolution. Analysis of the native Patterson map revealed a peak at 50% of the origin on the Harker section v = 0.5, suggesting twofold non-crystallographic symmetry parallel to the b crystallographic axis. As attempts to derivatize or grow selenomethionine-labelled protein crystals failed, in-drop proteolysis was used to produce new crystal forms. A trace amount of subtilisin Carlsberg was added to IpaD before sparse-matrix screening, resulting in the production of several new crystal forms. This approach produced SeMet-labelled crystals and diffraction data were collected to 3.2 A resolution. The SeMet crystals belong to space group C2, with unit-cell parameters a = 139.4, b = 45.0, c = 99.5 A, beta = 107.9 degrees . An anomalous difference Patterson map revealed peaks on the Harker section v = 0, while the self-rotation function indicates the presence of a twofold noncrystallographic symmetry axis, which is consistent with two molecules per asymmetric unit.

Antigens, Bacterial↗
PubMed1982

A 53-kilodalton protein common to chemically and virally transformed cells shows extensive sequence similarities between species.

A heat-stable DNA-binding protein with subunits of about 53 kilodaltons (kDal) was purified from two virally transformed human cell lines (Epstein-Barr virus-positive Raji and Namalwa) and two mouse tumor cell lines (methylcholanthrene-induced Meth A sarcoma and TA3 mammary carcinoma). All four 53kDal proteins showed closely related total amino acid compositions, similar peptide maps, and identical NH2-terminal amino acid sequences for 20 residues. These 53-kDal proteins are therefore evolutionarily highly conserved, independent of whether they originate from virally or chemically transformed cells. The NH2-terminal sequence and the protein chain as a whole are not hydrophobic; however, some unexpected residue distributions were observed. Comparisons with other proteins reveal no clear sequence similarity with known tumor antigen structures, homologous immunoglobulins, or some other proteins of known sequence. Epstein-Barr virus-determined nuclear antigen also appears to have a different NH2-terminal sequence. Thus, the results show that the 53-kDal proteins represent a unique protein type with little species variation; this finding suggests that these proteins must perform an important common function in different transformation systems.

Amino Acid Sequence↗
PubMed1994

Fine structure mapping and deletion analysis of the murine piebald locus.

piebald (s) is a recessive mutation that affects the development of two cell types of neural crest origin: the melanocytes, responsible for pigment synthesis in the skin, and enteric ganglia, which innervate the lower bowel. As a result, mice carrying piebald mutations exhibit white spotting in the coat and aganglionic megacolon. Previously the gene had been localized to the distal half of mouse chromosome 14. To determine its precise location relative to molecular markers, an intersubspecific backcross was generated. Two anchor loci of chromosome 14, slaty and hypogonadal, in addition to simple sequence length repeat markers, were used to localize s to a 2-cM interval defined by the markers D14Mit38 and D14Mit42. The molecular markers were also used to characterize nine induced s alleles. Three of these mutations exhibited no deletions or rearrangements of the flanking markers, whereas the other six had two or more of these markers deleted. The extent of the deletions was found to be consistent with the severity of the homozygous phenotype. The location of deletion breakpoints in the induced alleles, coupled with the recombination breakpoints in the backcross progeny, provide useful molecular landmarks to define the location of the piebald gene.

Animals↗
PubMed2006

In silico prediction of FVIII epitopes recognised by natural autoantibodies in polyvalent immunoglobulin concentrates.

Inhibitory antibodies directed against blood coagulation factor VIII (FVIII) impair FVIII replacement therapy, constituting a serious complication in haemophilic and autoimmune patients. Identifying B-cell FVIII epitopes and mapping them on the molecule remain important challenges. Using a combination of different algorithms, more than 30 hypothetical linear epitopes were predicted on the FVIII molecule surface. We selected several major predicted sequences, spanning all FVIII domains, for specific antibody induction in rabbits. All peptides tested successfully induced production of specific anti-FVIII rabbit antibodies, supporting the relevance of our approach. To investigate the presence of FVIII-reactive antibodies in the healthy donor population, a pooled fraction rich in all IgG subclasses was purified on peptide-Sepharose columns. Substantial amounts of Ig, specific for each FVIII peptide, were purified with yields ranging from 8 to 223 ng/mg immunoglobulins. Our results confirm the diversity of FVIII epitopes recognised by natural human anti-FVIII autoantibodies. All IgG subclasses were found in the affinity-isolated anti-peptide material, with overrepresentation of IgG2 and IgG4. Evidence was also found for new FVIII epitopes. Five human anti-peptide preparations displayed FVIII-neutralising activity, ranging from 1.3 to 5.3 BU/mg. Although the presence of naturally occurring anti-FVIII antibodies in healthy donors has been previously described, our methodology has allowed, for the first time, a fine mapping of several inhibitory and non-inhibitory epitopes. Our observations support the hypothesis that FVIII inhibitors in haemophilia A and autoimmune disease may originate from the proliferation of natural FVIII-specific B-cell clones.

Algorithms↗
PubMed1975

Raphe origin of serotonergic nerves terminating in the cerebral ventricles.

The dorsal and median raphe nuclei of the midbrain are known to contain the perikarya of origin of the major serotonergic (indoleamine) neurons projecting to the parenchyma of the forebrain. Lesions were placed in these nuclei to determine whether serotonin-containing nerve terminals in the cerebral ventricular system are also derived from the raphe nuclei. Brain tissue from control rats and rats 2-7 days after placement of raphe lesions was examined by fluorescence and electron microscopy. By the third day after lesion there was a marked reduction in the formaldehyde-induced fluorescence of supra-ependymal terminals. By the same time virtually all supra-ependymal terminals showed advanced degenerative changes as visualized by electron microscopy. There was a degeneration of supra-ependymal terminals in all parts of the cerebral ventricular system examined, including the epithalamic region (e.g., habenula and pineal recess; serotonin-containing terminals in the latter areas had previously been thought to arise from modified pinealocytes in the pineal recess). We conclude that most, if not all, supra-ependymal nerve terminals are derived from serotonergic cells of origin in the raphe nuclei.

Animals↗
PubMed1985

Cholinergic involvement in lateral hypothalamic rewarding brain stimulation.

Rats were implanted with stimulating electrodes in the lateral hypothalamus, and cannulae for chemical injections in the ventral tegmentum. Injections of atropine, a muscarinic antagonist, increased thresholds for self-stimulation in a dose-dependent fashion, without slowing bar pressing rates. Thresholds increased less for a self-stimulation site contralateral to the atropine injection. In a conditioned place preference test, the rats preferred compartments in which they received carbachol, a cholinergic agonist. Muscarinic receptors in ventral tegmentum therefore seem critical for medial forebrain bundle (MFB) reward. The possible cholinergic cells of origin are discussed.

Animals↗
PubMed2000

Mosaic rearrangement of chromosome 18: characterization by FISH mapping and DNA studies shows trisomy 18p and monosomy 18p both of paternal origin.

Structural abnormalities of chromosome 18p mainly consist of isochromosomes of the short arm, which result in tetrasomy 18p. Trisomy 18p is much rarer, and less well characterized. We report on a 12-year-old girl with minor facial anomalies, delayed development, abnormal hands, atopic dermatitis, and hearing loss. She was mosaic for two abnormal cell lines in peripheral blood. In 90% of cells, a dicentric chromosome with duplication of the whole short arm of chromosome 18 resulted in trisomy 18p; 10% of cells had monosomy 18p, arising from a t(14;18)(p11;q11). FISH mapping, with multiple region specific and locus specific probes from the short and long arm of chromosome 18, showed that the structure of the dicentric chromosome 18 was 18pter-->18q23::18q11-->18pter. DNA polymorphisms for chromosome 18 showed that the abnormalities of chromosome 18 were paternal in origin. Combining all results, we could link the trisomy 18p and monosomy 18p to a common origin via a complex series of events in an early mitosis.

Abnormalities, Multiple↗
PubMed2004

Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid.

Cmd 4 is a colcemid resistant beta-tubulin mutant of Chinese hamster ovary cells that exhibits hypersensitivity to paclitaxel and temperature sensitivity for growth. The mutant beta-tubulin allele in this cell line encodes a D45Y amino acid substitution that produces colcemid resistance by making microtubules more stable. By selecting revertants of the temperature sensitive and paclitaxel hypersensitive phenotypes, we have identified three cis-acting suppressors of D45Y. One suppressor, V60A, maps to the same region as the D45Y alteration, and a second suppressor, Q292H, maps to a distant location. Both appear to produce compensatory changes in microtubule assembly that counteract the effects of the original D45Y substitution. Consistent with this view, expression of the V60A mutation in transfected wild-type cells produced paclitaxel resistance and greatly decreased microtubule assembly. Additionally, it produced a paclitaxel-dependent phenotype in which cells grew normally in the presence, but not the absence, of the drug. The Q292H mutation caused even greater disassembly of microtubules such that cells were unable to proliferate when the transgene was expressed; but, unlike the V60A mutation, cell growth could not be rescued by paclitaxel. A third suppressor, A254V, maps to a region near the interface between alpha- and beta-tubulin that contains the colchicine binding site. Although it made transfected wild-type cells hypersensitive to colcemid, it did not affect paclitaxel or vinblastine sensitivity, nor did it reduce microtubule assembly. We suggest that this mutation acts by increasing tubulin's affinity for colcemid.

Alleles↗
PubMed2005

Neural crest origins of the neck and shoulder.

The neck and shoulder region of vertebrates has undergone a complex evolutionary history. To identify its underlying mechanisms we map the destinations of embryonic neural crest and mesodermal stem cells using Cre-recombinase-mediated transgenesis. The single-cell resolution of this genetic labelling reveals cryptic cell boundaries traversing the seemingly homogeneous skeleton of the neck and shoulders. Within this assembly of bones and muscles we discern a precise code of connectivity that mesenchymal stem cells of both neural crest and mesodermal origin obey as they form muscle scaffolds. The neural crest anchors the head onto the anterior lining of the shoulder girdle, while a Hox-gene-controlled mesoderm links trunk muscles to the posterior neck and shoulder skeleton. The skeleton that we identify as neural crest-derived is specifically affected in human Klippel-Feil syndrome, Sprengel's deformity and Arnold-Chiari I/II malformation, providing insights into their likely aetiology. We identify genes involved in the cellular modularity of the neck and shoulder skeleton and propose a new method for determining skeletal homologies that is based on muscle attachments. This has allowed us to trace the whereabouts of the cleithrum, the major shoulder bone of extinct land vertebrate ancestors, which seems to survive as the scapular spine in living mammals.

Amphibians↗
PubMed2005

Distal Chr 4 harbors a genetic locus (Gct1) fundamental for spontaneous ovarian granulosa cell tumorigenesis in a mouse model.

The spontaneous development of juvenile-onset ovarian granulosa cell tumors in mice of the SWXJ-9 recombinant inbred strain is a model for juvenile-type granulosa cell tumors that appear in very young girls. To expedite gene discovery in this mouse model of childhood cancer, we did a gene mapping study with the SWXJ-9 recombinant inbred strain and the evolutionarily divergent Mus musculus castaneus (CAST/Ei) strain as a mapping partner. Our mapping strategy focused on autosomal determinants of susceptibility with a backcross scheme that exploited a paternal, parent-of-origin effect for a X-linked gene (Gct4) that strongly supports granulosa cell tumor development. Of 1,968 backcross females examined, we detected 81 granulosa cell tumor-bearing animals and compared their allelic inheritance patterns to non-tumor-bearing siblings in a case-control analysis. The results of our study have confirmed an important locus on mouse chromosome (Chr) 4 (Gct1) and have revealed new loci for granulosa cell tumor susceptibility (Gct7-Gct9) on Chrs 1, 2, and 13 with susceptibility alleles contributed by the SWXJ-9 progenitor. Two novel gene-gene interactions supportive for granulosa cell tumor development were also observed between loci on Chrs 17 and 18 and loci on Chrs 2 and 10. Our data substantiate the evidence that Gct1 on Chr 4 is a fundamental oncogene for granulosa cell tumorigenesis in mice and has identified additional interacting autosomal loci that support tumor development.

Animals↗
PubMed2006

Locus coeruleus neurons originate in alar rhombomere 1 and migrate into the basal plate: Studies in chick and mouse embryos.

We investigated in the mouse and chick the neuroepithelial origin and development of the locus coeruleus (LoC), the most important noradrenergic neuronal population in the brain. We first studied the topography of the developing LoC in the hindbrain, using as markers the key noradrenergic marker gene Dbh and the transcription factors Phox2a and Phox2b (upstream of Dbh). In both mouse and chicken, LoC neurons first appear arranged linearly along the middle one-third of the alar plate of rhombomere 1 (r1), collinear to a reference ventricular longitudinal band that early on expresses Phox2a and Phox2b in the alar plate of r2 and later expands to r1. Double-labeling experiments with LoC markers (Dbh or Phox2a) and either alar (Pax7 and Rnx3) or basal (Otp) genetic markers suggested that LoC cells migrate from their origin in the alar plate to a final position in the lateral basal plate. To corroborate these suggestions experimentally and determine the precise origin of the LoC, we fate mapped the LoC in the chick at stage HH11 by using quail-chick homotopic grafts. The experimental results confirmed that the LoC originates in the alar plate throughout the rostrocaudal extent of r1 and ruled out a rostrocaudal translocation. They also corroborated a ventralward tangential migration of LoC cells into the lateral basal plate, where the postmigratory LoC primordium is located. Comparisons with neighboring alar r1-derived cell populations established that LoC neurons originate outside the cerebellum, in a matrix area intercalated dorsoventrally between the sources of the prospective vestibular and trigeminal columns.

Animals↗
PubMed1986

Kell blood group antigens are part of a 93,000-dalton red cell membrane protein.

Monospecific Kell blood group antibodies, of either human alloimmune or mouse monoclonal origin, react with a single surface-exposed protein of 93,000 daltons. Chymotryptic peptide maps of the 93,000-dalton protein isolated by antibodies of two different specificities (anti-K7 or anti-K14) indicate that Kell epitopes reside on the same protein. Kell protein is similar in size to band 3 protein but differs markedly in its tryptic and chymotryptic peptide maps, indicating that they are different proteins. In addition, sheep antibody to human band 3 does not react with Kell protein. Rabbit antibody to Kell protein reacts, by Western immunoblotting, with membrane proteins from Kell antigen positive red blood cells but not from those of a Ko (Kell null) cell. In intact red cells only a small portion of the Kell protein is available to lactoperoxidase-catalyzed iodination. Under nonreducing conditions Kell antigen is isolated not only as a 93,000-dalton protein but also as larger protein complexes ranging in size from above 200,000 to 115,000 daltons. Treatment of red cells with iodoacetamide, prior to isolation of Kell protein, reduces the amount of the very large complexes, but Kell protein occurs both as 115,000- and 93,000-dalton proteins.

Animals↗
PubMed2002

Change of mouse CD5(+) B1 cells to a macrophage-like morphology induced by gamma interferon and inhibited by interleukin-4.

The in vitro effects of gamma interferon (IFN-gamma) on the mouse CD5(+) B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-gamma, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-gamma. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-gamma-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-gamma and IL-4 exhibited contradictory actions on morphological change of CD5(+) B1 cells into macrophage-like cells. Differential regulation of CD5(+) B1 cells by IFN-gamma, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.

Animals↗
PubMed2002

Polar localization of the Escherichia coli oriC region is independent of the site of replication initiation.

The location of the origin-linked region of the Escherichia coli chromosome was analysed in strains lacking the core origin locus, oriC. In these strains, which initiate replication from F factors integrated at different locations around the chromosome, origin-linked DNA remains localized near the cell poles, as in wild-type cells. In contrast, minichromosomes containing 7 kb of chromosomal DNA including oriC are generally excluded from the ends of the cell. Thus, we propose that positioning of the wild-type origins at the poles is not a function of their order of replication but a sequence-specific phenomenon. It is proposed that there are centromere-like sequences, bordering the wild-type origin of replication, which are used by host mechanisms to direct the proper placement of the origin region of the chromosome. This function, combined with other host processes, may assure efficient segregation of the E. coli chromosome.

Cell Polarity↗
PubMed1985

Enhancer-facilitated expression of a human H4 histone gene.

Cultured mammalian cells were transfected with a recombinant human H4 histone gene. S1 nuclease mapping of cellular RNAs from transfected cells revealed: (i) correct initiation of transcription at the cap site, with some transcripts originating from other sites in the 5' flanking region of this H4 gene; (ii) cis-linkage of an SV-40 transcriptional enhancer element upstream of the H4 5'-flanking region resulted in about a 50-fold increase in the level of correctly initiated H4 mRNA and (iii) in a heterologous murine system stability of human H4 mRNAs was apparently sensitive to inhibition of DNA-synthesis by hydroxyurea. Our results suggest that certain sequences required for the initiation of a human H4 histone gene transcript reside within the 210 nucleotides immediately upstream from the cap site and that the level of expression is influenced by the introduction of an enhancer element.

Animals↗
PubMed1992

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.

Blotting, Southern↗
PubMed1988

[Differentiation of the neural crest. Influence of growth factors].

The peripheral nervous system (PNS) is derived from a transitory embryonic structure, the neural crest with an additional contribution of the cephalic ectodermal placodes to the sensory ganglia of certain cranial nerves. In vivo grafting experiments, taking advantage of the quail/chick marker system, allowed the fate map of the neural crest to be constructed and the precise level of origin along the neuraxis of the component cells of PNS ganglia to be defined. It appeared that the neural crest is regionalized in different areas from which a limited number of phenotypes arise. However, if the position of neural crest fragments is experimentally changed prior to the onset of migration, it turns out that virtually all the cell types represented in PNS ganglia can arise from any level of the neural crest, provided it is transplanted into the appropriate level of the neuraxis. This means that the microenvironment into which the neural crest cells migrate plays a critical role in directing their differentiation. Further in vivo studies along with in vitro clonal analysis of neural crest developmental potencies revealed that, during the migratory phase, the crest cells are already highly heterogeneous in their state of commitment. When they reach the sites of gangliogenesis, they exhibit different requirements for growth and differentiation. Our current studies are aimed at identifying the factors that, in spinal and sympathetic ganglia respectively, trigger the differentiation of sensory and adrenergic sympathetic neurons. The brain-derived neurotrophic factor (BDNF) and insulin (and/or IGF1) have been shown to play a role in these processes.

Animals↗