Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC 2.4.1.17; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (P = 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%, P < 0.0001; variant homozygotes: 50% vs. 14.7%, P = 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%, P = 0.006) or variant homozygous (50%, P = 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.

Third-generation synchrotron x-ray diffraction of 6-μm crystal of raite, ≈Na3Mn3Ti0.25Si8O20(OH)2⋅10H2O, opens up new chemistry and physics of low-temperature minerals

The crystal structure of raite was solved and refined from data collected at Beamline Insertion Device 13 at the European Synchrotron Radiation Facility, using a 3 × 3 × 65 μm single crystal. The refined lattice constants of the monoclinic unit cell are a = 15.1(1) Å; b = 17.6(1) Å; c = 5.290(4) Å; β = 100.5(2)°; space group C2/m. The structure, including all reflections, refined to a final R = 0.07. Raite occurs in hyperalkaline rocks from the Kola peninsula, Russia. The structure consists of alternating layers of a hexagonal chicken-wire pattern of 6-membered SiO4 rings. Tetrahedral apices of a chain of Si six-rings, parallel to the c-axis, alternate in pointing up and down. Two six-ring Si layers are connected by edge-sharing octahedral bands of Na+ and Mn3+ also parallel to c. The band consists of the alternation of finite Mn–Mn and Na–Mn–Na chains. As a consequence of the misfit between octahedral and tetrahedral elements, regions of the Si–O layers are arched and form one-dimensional channels bounded by 12 Si tetrahedra and 2 Na octahedra. The channels along the short c-axis in raite are filled by isolated Na(OH,H2O)6 octahedra. The distorted octahedrally coordinated Ti4+ also resides in the channel and provides the weak linkage of these isolated Na octahedra and the mixed octahedral tetrahedral framework. Raite is structurally related to intersilite, palygorskite, sepiolite, and amphibole.

Chimeric Cytokine Receptor Can Transduce Expansion Signals in Interleukin 6 Receptor α (IL-6Rα)-, IL-11Rα-, and gp130-low to -negative Primitive Hematopoietic Progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin−), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin−Sca-1+c-kit+ progenitors and increased either mixed colony-forming unit or cobblestone area–forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin−Sca-1+c-kit+CD34− further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Rα-, IL-11Rα-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.

Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an α-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with “choices” during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual α-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: Are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

A carbonic anhydrase from the nacreous layer in oyster pearls.

It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

The [(G/C)3NN]n motif: a common DNA repeat that excludes nucleosomes.

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3 NN3']n motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)48 block is 4.9 +/- 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and approximately 78-fold less efficient than a similar length (CTG)n sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of "TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Layer-specific programs of development in neocortical projection neurons.

How are long-range axonal projections from the cerebral cortex orchestrated during development? By using both passively and actively transported axonal tracers in fetal and postnatal ferrets, we have analyzed the development of projections from the cortex to a number of thalamic nuclei. We report that the projections of a cortical area to its corresponding thalamic nuclei follow highly cell-specific programs of development. Axons from cells in the deepest layers of the cerebral cortex (layer 6 and superficial subplate neurons) appear to grow very slowly and be delayed for several weeks in the cerebral white matter, reaching the thalamus over a protracted period. Neurons of layer 5, on the other hand, develop their projections much faster; despite being born after the neurons of deeper layers, layer 5 neurons are the first to extend their axons out of the cortical hemisphere and innervate the thalamus. Layer 5 projections are massive in the first postnatal weeks but may become partly eliminated later in development, being overtaken in number by layer 6 cells that constitute the major corticothalamic projection by adulthood. Layer 5 projections are area-specific from the outset and arise as collateral branches of axons directed to the brainstem and spinal cord. Our findings show that the early development of corticofugal connections is determined not by the sequence of cortical neurogenesis but by developmental programs specific for each type of projection neuron. In addition, they demonstrate that in most thalamic nuclei, layer 5 neurons (and not subplate or layer 6 neurons) establish the first descending projections from the cerebral cortex.

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Corneal Biomechanics, Retinal Nerve Fiber Layer, and Optic Disc in Children

Purpose: To evaluate the possible associations between corneal biomechanical parameters, optic disc morphology, and retinal nerve fiber layer (RNFL) thickness in healthy white Spanish children. Methods: This cross-sectional study included 100 myopic children and 99 emmetropic children as a control group, ranging in age from 6 to 17 years. The Ocular Response Analyzer was used to measure corneal hysteresis (CH) and corneal resistance factor. The optic disc morphology and RNFL thickness were assessed using posterior segment optical coherence tomography (Cirrus HD-OCT). The axial length was measured using an IOLMaster, whereas the central corneal thickness was measured by anterior segment optical coherence tomography (Visante OCT). Results: The mean (±SD) age and spherical equivalent were 12.11 (±2.76) years and −3.32 (±2.32) diopters for the myopic group and 11.88 (±2.97) years and +0.34 (±0.41) diopters for the emmetropic group. In a multivariable mixed-model analysis in myopic children, the average RNFL thickness and rim area correlated positively with CH (p = 0.007 and p = 0.001, respectively), whereas the average cup-to-disc area ratio correlated negatively with CH (p = 0.01). We did not observe correlation between RNFL thickness and axial length (p = 0.05). Corneal resistance factor was only positively correlated with the rim area (p = 0.001). The central corneal thickness did not correlate with the optic nerve parameters or with RNFL thickness. These associations were not found in the emmetropic group (p > 0.05 for all). Conclusions: The corneal biomechanics characterized with the Ocular Response Analyzer system are correlated with the optic disc profile and RNFL thickness in myopic children. Low CH values may indicate a reduction in the viscous dampening properties of the cornea and the sclera, especially in myopic children.

South America, 1807 (Image 6 of 7) (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Colombia Prima or South America : in which it has been attempted to delineate the extent of our knowledge of that continent, extracted chiefly from the original manuscript maps of ... Pinto, likewise fom those of João Joaquin da Rocha, João da Costa Ferreira ... Francisco Manuel Sobreviela &c. and from the most authentic edited accounts of those countries, digested & constructed by ... Louis Stanislas D'Arcy Delarochette. It was published by William Faden geographer to His Majesty and to His Royal Highness the Prince of Wales in June 4th, 1807. Scale [ca. 1:3,000,000]. This layer is image 6 of 7 total images of the eight sheet map, representing the western portion of the map.The image inside the map neatline is georeferenced to the surface of the earth and fit to the South America Lambert Conformal Conic projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, cities and other human settlements, roads, territorial boundaries, shoreline features, mines, tribes, and more. Relief shown by hachures. Includes notes.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.