Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Sequence and structural relationships in the cytokine family

The sequences of nine different cytokines, growth hormone, and prolactin have been aligned and their secondary structure predicted. The alignment reveals that each exon has a characteristic sequence pattern shared by all cytokines. The most striking sequence similarity is observed in exon 4, where the residue pair Phe-Leu is conserved in many cytokines. In addition, there are discreet homologous regions between two specific growth factors, including a high degree of homology between granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). The secondary structure analysis predicts that exon 3 of all cytokines has an antiparallel helix-turn-helix motif, which is likely to form the central helical segments of a four -helical bundle-type structure. Based on the secondary structure and the disulfidebonding pattern, the topological connectivity for a number of cytokines has been predicted.     

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).     

Cellular oxidative stress response mediates radiosensitivity in Fus1-deficient mice

Mechanism of radiosensitivity of normal tissues, a key factor in determining the toxic side effects of cancer radiotherapy, is not fully understood. We recently demonstrated that deficiency of mitochondrial tumor suppressor, Fus1, increases radiosensitivity at the organismal, tissue and cellular levels. Since Fus1-deficient mice and cells exhibit high levels of oxidative stress, we hypothesized that dysregulation of cellular antioxidant defenses may contribute to the increased radiosensitivity. To address this potential mechanism, we treated the Fus1 KO mice with an inhibitor of pathogenic oxidative reactions, pyridoxamine (PM). Treatment with PM ameliorated IR-induced damage to GI epithelium of Fus1 KO mice and significantly increased the survival of irradiated mice. In cultured Fus1 KO epithelial cells, IR-induced oxidative stress was enhanced because of inadequate cellular antioxidant defenses, such as low levels and/or activities of cytochrome C, Sod 2 and STAT3. This resulted in dysregulation of IR-induced DNA-damage response and DNA synthesis. Treatment of Fus1 KO cells with PM or Sod 2 mimetic Tempol normalized the oxidative stress response, thus compensating to a significant degree for inadequate antioxidant response. Our findings using Fus1 KO radiosensitive mice suggest that radiosensitivity is mediated via dysregulation of antioxidant response and defective redox homeostasis.Normal tissue sensitivity to ionizing radiation (IR) limits the therapeutic dose that can be delivered to the tumor and is responsible for early and late side effects. Minimizing the toxic effects of IR in normal cells would significantly alleviate the side effects and improve the outcome of radiotherapy. However, mechanisms regulating the response of normal tissues to IR are still not completely understood. Ionizing radiation is a strong inducer of reactive oxygen species (ROS) that are produced mostly by mitochondria.¹ Overproduction of ROS at the time of irradiation causes mitochondrial dysfunction followed by an oxidative damage to mitochondrial DNA and proteins.² It has also been suggested that ROS generated by mitochondria contribute to genomic instability after radiation exposure.²We previously demonstrated that mitochondrial tumor suppressor Fus1 can modulate radiosensitivity of normal gastrointestinal (GI) tract epithelium,³ one of the primary targets of ionizing radiation during whole body exposure or pelvic radiotherapy.^{4, 5, 6} Given that Fus1 is involved in the regulation of mitochondrial homeostasis including generation of ROS^{7, 8} and that levels of Fus1 expression vary significantly in individual humans,^{9, 10} these findings may provide mechanistic insights into IR toxicity in normal tissues. Moreover, expression levels of Fus1 may predict individual susceptibility to radiation toxicity. To determine the mechanisms whereby Fus1 modulates cellular radiosensitivity, we utilized Fus1 KO mouse model and inhibitors of oxidative pathways with different mechanisms of actions, pyridoxamine (PM) and Tempol.Fus1 KO mice are characterized by mitochondrial dysfunction and elevated oxidative stress.^{8, 11} Upon whole body irradiation (WBI), these mice exhibit increased mortality because of GI toxicity driven by accelerated apoptosis and untimely re-entry into cell cycle leading to GI crypt epithelial cell death and diminished crypt regeneration.³ Suggested causative mechanisms include dysregulation of cell cycle, apoptotic signaling, DNA repair and oxidative stress response.³ Since ROS generation occurs immediately upon irradiation¹² and high ROS levels and oxidative stress are the main features of mitochondrial dysfunction in Fus1 KO mice,^{8, 11} we hypothesized that amelioration of oxidative stress may abrogate increased radiosensitivity of Fus1 KO tissues, thus improving crypt regeneration and animal survival.Pyridoxamine treatment ameliorates pathogenic oxidative pathways in diabetes and other pro-oxidative stress conditions, including following exposure to ionizing radiation.^{13, 14} PM has been shown to scavenge and inhibit the production of toxic ROS and carbonyl species, which also are major damaging factors in irradiated biological tissues.¹⁴ Tempol is a cell membrane-permeable amphilite that dismutates superoxide catalytically and facilitates hydrogen peroxide metabolism by catalase-like actions.¹⁵In the present study, we demonstrated that treatment with PM ameliorated IR-induced damage to GI epithelium of Fus1 KO mice and significantly increased the survival of these mice upon irradiation. Cell culture experiments showed that loss of Fus1 protein enhanced IR-induced oxidative stress because of inadequate cellular antioxidant defenses. This resulted in dysregulation of IR-induced DNA-damage response and DNA synthesis. Treatment of Fus1 KO cells with PM or Tempol normalized the oxidative stress response, thus compensating to a significant degree for the lack of Fus1. These data suggest that mechanisms of radiosensitivity can be determined by dysregulation of antioxidant response and defective redox homeostasis.     

The Midline Protein Regulates Axon Guidance by Blocking the Reiteration of Neuroblast Rows within the Drosophila Ventral Nerve Cord

Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.     

Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis

Toll-like receptors (TLRs) play a central role in the innate immune response by recognizing conserved structural patterns in a variety of microbes. TLRs are classified into six families, of which TLR7 family members include TLR7, 8, and 9, which are localized to endolysosomal compartments recognizing viral infection in the form of foreign nucleic acids. In our current study, we focused on TLR8, which has been shown to recognize different types of ligands such as viral or bacterial ssRNA as well as small synthetic molecules. The primary sequences of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the TLR8 pathway is species-specific. Moreover, the factors underlying the receptor's species-specificity remain unknown. To this end, comparative homology modeling, molecular dynamics simulations refinement, automated docking and computational mutagenesis studies were employed to probe the intermolecular interactions between this anti-viral compound and TLR8. Furthermore, comparative analyses of modeled TLR8 (rodent and non-rodent) structures have shown that the variation mainly occurs at LRR14-15 (undefined region); hence, we hypothesized that this variation may be the primary reason for the exhibited species-specificity. Our hypothesis was further bolstered by our docking studies, which clearly showed that this undefined region was in close proximity to the ligand-binding site and thus may play a key role in ligand recognition. In addition, the interface between the ligand and TLR8s varied depending upon the amino acid charges, free energy of binding, and interaction surface. Therefore, our current work provides a hypothesis for previous in vivo studies in the context of TLR signaling.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.