Localization and expression of a 70 kDa protein in goat spermatozoa having Na+,K+-ATPase inhibitory and arylsulphatase A activities

We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.     

Spatio-Temporal Plasticity in Chromatin Organization in Mouse Cell Differentiation and during Drosophila Embryogenesis

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells, suggesting possible mechanisms to alter gene expression programs, yet the physical basis of this organization is unclear. In this article, we contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem cells and terminally differentiated primary mouse embryonic fibroblasts. Live-cell confocal tracking of nuclear lamina evidences highly flexible nuclear architecture within embryonic stem cells as compared to primary mouse embryonic fibroblasts. These cells also exhibit significant changes in histone and heterochromatin binding proteins correlated with their distinct epigenetic signatures as quantified by immunofluorescence analysis. Further, we follow histone dynamics during the development of the Drosophila melanogaster embryo, which gives an insight into spatio-temporal evolution of chromatin plasticity in an organismal context. Core histone dynamics visualized by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy within the developing embryo, revealed an intriguing transition from plastic to frozen chromatin assembly synchronous with cellular differentiation. In the embryo, core histone proteins are highly mobile before cellularization, actively exchanging with the pool in the yolk. This hyperdynamic mobility decreases as cellularization and differentiation programs set in. These findings reveal a direct correlation between the dynamic transitions in chromatin assembly with the onset of cellular differentiation and developmental programs.     

Structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass

Ball-milled alcohol-insoluble residue (AIR) was prepared from switchgrass (Panicum virgatum var Alamo) and sequentially extracted with 50 mM ammonium oxalate buffer, 50 mM sodium carbonate, 1 M KOH containing 1% NaBH₄, and 4 M KOH containing 1% NaBH₄. Arabinoxylan was the most abundant component of the 1 M KOH-extracted fraction, which was treated with endoxylanase to generate oligosaccharides. Gel-permeation chromatography of these oligosaccharides produced three size-homogeneous oligosaccharide fractions with molecular weights of 678, 810, and 1074 Da, corresponding to 5, 6, and 8 pentose units, respectively. Detailed structural analysis of these oligosaccharides was performed using methylation analysis, multiple-step mass spectrometry (ESIMSⁿ), and 1D and 2D NMR spectroscopy. The preferred gas-phase fragmentation pathways were identified for these oligosaccharides, providing extensive sequence information that was completely consistent with structures determined by ab initio NMR analysis. These results demonstrate the high information content of ESIMSⁿ analysis when applied to cell-wall-derived oligosaccharides and provide standard data that will facilitate the analysis of cell-wall polysaccharide fragments with a sensitivity that is sufficient for the analysis of samples obtained from dissected tissues as well as other small samples.     

Lysosome rich cells contain the lytic activity of lymphokine-activated killer cell populations

Summary Little is known regarding the effectors of lymphokine-activated killer activity. Lysosomotropic agents such as quinacrine can be used to positively sort for lysosome rich cells in natural killer (NK) cell populations. We therefore decided to use this agent to sort lymphokine-activated killer (LAK) cells to characterize their lysosomal content. We found that the positively sorted population contained all the LAK activity, i.e., lysis of NK-resistant tumor cells (B16 melanoma cell line), with the negatively sorted cells having no killing activity. Therefore separation of interleukin-2-incubated cells for LAK activity could be accomplished using sorting after quinacrine staining. The treatment of positively sorted LAK cell populations with L-leucine methyl ester, a lysosomotropic dye which inhibits killing by lysosome rich cells, caused abrogation of killing of the B16 tumor by the treated populations. Single cell conjugate assays were also done on these sorted cells, with positively sorted cells forming the highest and negatively sorted cells the lowest percent of conjugates. Our data therefore indicates the important role of lysosome rich cells in the LAK cell population in the murine system.This work was supported by NIH grants R01 CA42962 and K04 CA0122, and by intramural funds from the Norris Cancer Center     

Stimulation of nitric oxide synthesis and protective role of insulin in acute thrombosis in vivo

Administration of physiologic amounts of insulin in mice (200 microunits/g body weight) resulted in 9 fold increase of basal nitric oxide level from 0.51+/-0.1224 nmol/ml (mean+/-SD, n=12) to 4.45+/-0.645 nmol/ml after 30min of the injection of the hormone. Since NO is a potent inhibitor of platelet aggregation both in vitro and in vivo, we tested the possibility whether the administration of the hormone would result in the in vivo inhibition of thrombosis through the increase of NO level in the circulation. It was found that administration of insulin (200 microunits/g body weight) in mice protected >90%(p<0.00001, n=500) of these animals from death due to thrombosis in the coronary arteries induced by ADP injection in the heart. This effect of insulin in vivo was found to be directly related to the hormone induced increase of NO level in the system. The thromboprotective effect of insulin could not be achieved by using either prostacyclin, a well known antithrombotic agent or its stable probe prostaglandin E1 instead of insulin. The efficacy of insulin was neither related to the blood glucose level nor was the consequence of the hypoglycemic effect of the hormone. In contrast, inhibition of insulin induced increase of NO level resulted in the complete loss of the thromboprotective effect of the hormone. These results suggest that insulin besides being a hypoglycemic hormone could also be a potent antithrombotic humoral factor.     

CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.