Ethnic-specific allelic variation within the D1Z2 locus

DNAs from 122 individuals representing 5 ethnic groups (Black, Chinese, Japanese, Caucasian and Melanesian) were analyzed for restriction fragment length polymorphisms (RFLPs) with a hypervariable repeated sequence located uniquely on chromosome 1 (hMF No.1; is a component of the D1Z2 locus). When human genomic DNA is digested with a variety of enzymes (TaqI, EcoRI, SinI, PstI, HaeIII) the hMF No.1 probe reveals multiple RFLPs. Ethnic group differences were found in the frequencies of specific EcoRI bands. The most striking ethnic group variation was the presence of a unique fragment amongst the Japanese.     

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.     

Spore Refractility in Variants of Bacillus cereus Treated with Actinomycin D

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)1, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. (45)Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of (45)Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.     

Novel Vascular Endothelial Growth Factor D Variants with Increased Biological Activity

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D^ΔNΔC) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D^ΔNΔC, the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D^ΔNΔC. The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D^ΔNΔC. More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D^ΔNΔC and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D^ΔNΔC. The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D^ΔNΔC described here are potential agents for therapeutic applications, where induction of vascular formation is required.Vascular endothelial growth factors (VEGFs)3 are considered as key growth factors inducing angiogenesis and lymphangiogenesis during embryogenesis as well as maintaining vasculature during adulthood. Their abnormal expression is found in pathological conditions such as cancer and retinopathies (1). Five mammalian VEGFs, VEGF-A, -B, -C, -D and placenta growth factor (PlGF), are known (2) as well as Orf virus-derived VEGF-E proteins (3) and multiple homologues from snake venoms (VEGF-Fs) (4). Several members of the VEGF family exist as different isoforms, either as a result of the alternative splicing of their mRNAs or due to proteolytic processing. These forms vary in their specificities and affinities to three main VEGF receptors, co-receptors such as neuropilins and heparan sulfate proteoglycans and other components of the extracellular matrix, translating into different biological effects (5).VEGFR-2 is an important receptor regulating vasculogenesis and angiogenesis. It is mainly expressed on endothelial cells, but expression is also found in several other cell types (6). Mammalian VEGFR-2 ligands include VEGF-A (7), VEGF-C (8), and VEGF-D (9). In addition to VEGFR-2, VEGF-C and VEGF-D are ligands for VEGFR-3, which mainly mediates lymphangiogenesis in adults but also participates in the formation of blood vessels during embryogenesis (2).Because of their importance in angiogenesis, VEGFs have been suggested as potential therapeutic agents in different pathological conditions to improve compromised blood flow. Studies aiming at inducing angiogenesis in vivo have been performed by introducing VEGFs to tissues either directly as recombinant proteins (10) or using gene therapy vectors (11). Findings from several laboratories have shown that VEGFs have strong angiogenic activity in vivo, and they could be used for the treatment of conditions like lower limb ischemia and ischemic coronary artery disease. The short in vivo half-life of these growth factors and the requirement for sustained angiogenic stimulus makes gene therapy a preferred option. Of the VEGFs, VEGF-A and the mature form of VEGF-D (VEGF-D^ΔNΔC, see below) are the strongest agents to induce vascular formation (12, 13).VEGFs share structural similarity with platelet-derived growth factors. Together they are classified as the VEGF/platelet-derived growth factor family, belonging to a larger family of cystine knot growth factors. The members of this family share a cystine knot motif, which is found in many extracellular proteins and is conserved among numerous species (14). Characteristic of the cystine knot proteins is that they contain a conserved structure of antiparallel β-sheets connected by three disulfide bonds. Typically cystine knot growth factors form dimers, which within the VEGF/platelet-derived growth factor family are often linked by intersubunit disulfide bonds. The crystal structures have been solved for VEGF-A (15), PlGF (16), VEGF-B (17), VEGF-E (18), and two snake venom VEGF-Fs, vammin and VR-1 (19).There are currently no published structures of VEGF-C or VEGF-D. They can be divided into their own subfamily based on sequence similarity and several characteristic features; 1) they are the only VEGFs that bind to VEGFR-3, 2) they are expressed as long precursor forms having poor receptor binding affinities, and 3) they require proteolytic processing at their N-terminal and C-terminal ends to become more active. In contrast to other members of the VEGF family, the mature, proteolytically processed ΔNΔC-forms of VEGF-C and VEGF-D exist predominantly as non-covalently bound dimers, even though they have the conserved cysteine residues that form the intersubunit disulfide bonds in other VEGFs (8, 20). However, both VEGF-C and VEGF-D also have an additional cysteine residue located at the dimer interface (8, 20). Mutation of this residue in VEGF-C only minimally altered the receptor binding affinity (21), but it stabilized the dimer structure (56).In the current study we investigated the importance of residues at the subunit interface for the function of VEGF-D^ΔNΔC. We built homology models of VEGF-D^ΔNΔC and used alanine scanning and site-specific mutagenesis as well as tested the biological activity of various mutated forms of VEGF-D^ΔNΔC. Our study revealed that the conserved cysteine residues (Cys-44 and Cys-53), which are known to form intersubunit disulfide bridges in other VEGFs, were essential for the activity of the recombinant VEGF-D^ΔNΔC. Furthermore, the monomer to dimer ratio of VEGF-D^ΔNΔC could be regulated by mutagenesis. In addition, it was found that replacement of the “extra” cysteine (Cys-25) by various amino acids, preferably Ile, Leu, or Val, actually enhanced the activity of VEGF-D^ΔNΔC. This was at least partially due to increased stability of the protein.     

Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity

Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn₄CaO₅ core of the water-oxidizing complex (WOC). Most cyanobacteria have 3–5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O₂ yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O₂ than either cyanobacterial D1 isoform. D1:2-PSII makes more O₂ per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S₃ state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.     

Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA-bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.     

Effects of GWAS-Associated Genetic Variants on lncRNAs within IBD and T1D Candidate Loci

Long non-coding RNAs are a new class of non-coding RNAs that are at the crosshairs in many human diseases such as cancers, cardiovascular disorders, inflammatory and autoimmune disease like Inflammatory Bowel Disease (IBD) and Type 1 Diabetes (T1D). Nearly 90% of the phenotype-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWAS) lie outside of the protein coding regions, and map to the non-coding intervals. However, the relationship between phenotype-associated loci and the non-coding regions including the long non-coding RNAs (lncRNAs) is poorly understood. Here, we systemically identified all annotated IBD and T1D loci-associated lncRNAs, and mapped nominally significant GWAS/ImmunoChip SNPs for IBD and T1D within these lncRNAs. Additionally, we identified tissue-specific cis-eQTLs, and strong linkage disequilibrium (LD) signals associated with these SNPs. We explored sequence and structure based attributes of these lncRNAs, and also predicted the structural effects of mapped SNPs within them. We also identified lncRNAs in IBD and T1D that are under recent positive selection. Our analysis identified putative lncRNA secondary structure-disruptive SNPs within and in close proximity (+/−5 kb flanking regions) of IBD and T1D loci-associated candidate genes, suggesting that these RNA conformation-altering polymorphisms might be associated with diseased-phenotype. Disruption of lncRNA secondary structure due to presence of GWAS SNPs provides valuable information that could be potentially useful for future structure-function studies on lncRNAs.     

Morphological Variants of Coliphage P1

Lysates of P1 from all hosts tested contained at least three morphological variants with respect to head size. These were termed "big" (P1B), "small" (P1S), and "minute" (P1M). Since successive clonings of plaques isolated on many different hosts failed to change the proportions of the variants, we concluded that the production of variants was a function of the P1 genome rather than that of the host. In the electron microscope, the heads appeared to be icosadeltahedra, having face-to-face head diameters of 86 +/- 2 nm, 65 +/- 2 nm, and 47 +/- 2 nm. Assuming the head capsids to be composed of the same protein subunits, these diameters were compatible with T = 16, 9, and 4 with a lattice constant (intercapsomere distance) of 12 to 13 nm. The tails of all variants were morphologically indistinguishable. Each consisted of a hollow tail tube surrounded by a contractile sheath attached to the head by means of a "head-neck connector" which could be a specialized vertex capsomere. In CsCl gradients, a number of bands were observed. One band contained the majority of P1B particles and 99% of the plaque-forming units. Two other bands contained P1S particles whose densities suggested a content of about 40 and 60% of the complete P1B genome. The less dense of these two bands also contained defective P1B particles with a calculated content of only 60% of the complete genome. The P1S particles tested injected their deoxyribonucleic acid (DNA) into host cells and killed them. Genetic markers contained in this band could be rescued by infectious P1B particles, confirming the evidence of Ikeda and Tomizawa that this fraction contains P1 DNA.     

Functional Genetic Variants of TNFSF15 and Their Association with Gastric Adenocarcinoma: A Case-Control Study

The purpose of this study was to identify functional genetic variants in the promoter of tumor necrosis factor superfamily member 15 (TNFSF15) and evaluate their effects on the risk of developing gastric adenocarcinoma. Forty DNA samples from healthy volunteers were sequenced to identify single nucleotide polymorphisms (SNPs) in the TNFSF15 promoter. Two TNFSF15 SNPs (−358T>C and −638A>G) were identified by direct sequencing. Next, genotypes and haplotypes of 470 gastric adenocarcinoma patients and 470 cancer-free controls were analyzed. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Serologic tests for Helicobacter pylori infection were measured by enzyme-linked immuno-sorbent assay (ELISA). Subjects carrying the TNFSF15 −358CC genotype were at an elevated risk for developing gastric adenocarcinoma, compared with those with the −358TT genotype (OR 1.42, 95% CI, 1.10 to 2.03). H. pylori infection was a risk factor for developing gastric adenocarcinoma (OR 2.31, 95% CI, 1.76 to 3.04). In the H. pylori infected group, subjects with TNFSF15 −358CC genotype were at higher risks for gastric adenocarcinoma compared with those carrying −358TT genotype (OR: 2.01, 95%CI: 1.65 to 4.25), indicating that H. pylori infection further influenced gastric adenocarcinoma susceptibility. The −358 T>C polymorphism eliminates a nuclear factor Y (NF-Y) binding site and the −358C containing haplotypes showed significantly decreased luciferase expression compared with −358T containing haplotypes. Collectively these findings indicate that functional genetic variants in TNFSF15 may play a role in increasing susceptibility to gastric adenocarcinoma.     

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.     

Immunoprecipitation of labeled antigens with Eupergit C1Z

Eupergit C1Z, a nonporous sedimentable acrylic polymer is shown to be a convenient solid support for isolation of antigens with high purity from small amounts of detergent-solubilized cells. Several applications of Eupergit C1Z coupled with antibodies and anti-antibodies are described, and compared with two other methods of immunoprecipitation.     

Regulation of expression of phospholipase D1 and D2 by PEA-15, a novel protein that interacts with them

Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.     

Occurrence of multiple sexual chromosomes (XX/XY1Y2 and Z1Z1Z2Z2/Z1Z2W1W2) in catfishes of the genus Ancistrus (Siluriformes: Loricariidae) from the Amazon basin

Loricariid catfishes show a predominance of homomorphism in sex chromosomes, but cases of simple and multiple systems were also found. Here we describe two cases of multiple sex chromosome systems in loricariids from Brazilian Amazonia. Males of Ancistrus sp.1 "Balbina" have a modal number of 2n = 39 chromosomes, fundamental number (FN) of 78, and karyotypic formula of 27 m + 10 sm + 2 st; females have 2n = 38 chromosomes, FN = 76, and 26 m + 10 sm + 2 st. Ancistrus sp.2 "Barcelos" has 2n = 52 chromosomes for both sexes, FN = 80 for males and FN = 79 for females. Karyotypic formula is 12 m + 12 sm + 4 st + 24a for males and 11 m + 12 sm + 4st + 25a for females. The two species show different arrangements of constitutive heterochromatin blocks, which are coincident with NORs and absent in sex chromosomes. We suggest a XX/XY(1)Y(2) mechanism for Ancistrus sp.1 "Balbina", and a Z(1)Z(1)Z(2)Z(2)/Z(1)Z(2)W(1)W(2) mechanism for Ancistrus sp.2 "Barcelos". The XX/XY(1)Y(2) mechanism here reported is the second known occurrence of this type of multiple sex chromosomes for Loricariidae and the third for Neotropical fishes; the mechanism Z(1)Z(1)Z(2)Z(2)/Z(1)Z(2)W(1)W(2) represents the first record among fishes. The presence of different sex chromosome systems in Ancistrus indicates a probable independent origin and suggests that the differentiation of sex chromosomes is evolutionarily recent among species in this genus.     

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2–Bub2–Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.