Cytochemical and immunocytochemical studies of the localization of histones and protamine-type proteins in spermatids of Chara vulgaris and Chara tomentosa

Spermiogenesis in Chara algae, which has been divided into 10 phases (sp I-X), is similar to spermiogenesis in animals. The most important process during spermiogenesis in animals is remodeling of chromatin leading to "sleeping genome", being the result the exchange of histone proteins into protamine-like proteins. Cytochemical studies showed in both Chara species (C. vulgaris, C. tomentosa) that at spI-IV phases only histones were present, at spV-VIII phases--the amount of nuclear protamine-type proteins progressively increased and that of histones decreased while at spIX-X only pro-tamine-type proteins were present. This was also confirmed with capillar electrophoresis. In order to localize more precisely both histones and protamines the immunocytochemical studies with the use of anti-protamine antibodies (protamine-type proteins were obtained from C. tomentosa antheridia) and anti-histone H3 antibodies, have been carried out. More specific immunocytochemical studies confirmed cytochemical results including the exchange of histones into protamine-type during spermiogenesis (spV-VIII) in both Chara species. At phase V spermiogenesis these strong strand-like anti-protamine signals were observed in cytoplasm which might suggest that protamine synthesis took place in ER.     

Role of proteolysis in caspase-8 activation and stabilization

Caspase-8 is an apoptotic protease that is activated at the cytosolic face of the cell membrane. Activation relies on adaptor-induced dimerization of monomeric caspase-8 and is followed by specific limited autoproteolysis of the linker which separates the two subunits of the catalytic domain. However, the role of this autoproteolysis, which directly activates executioner caspases-3 and -7, is unknown for the apical caspase-8. We have generated linker mutants of caspase-8 that can be proteolyzed in a controlled manner by thrombin or tobacco etch mosaic virus protease, and we use these to define the role of proteolysis in the activation and stability of the enzyme. We show that proteolysis is insufficient for generating enzymatic activity in recombinant caspase-8. Kinetic activation studies using Hoffmeister salts demonstrate that activation is the result of caspase dimerization. However, linker proteolysis significantly enhances the equilibrium for caspase-8 dimerization, thereby increasing the stability of the dimer. Kinetic and fluorescence measurements demonstrate that caspase-8 activation by Hoffmeister salts is at least a two-step event, with the required step being dimerization, followed by an intramolecular event that further stabilizes the catalytic conformation. Autoproteolysis of caspase-8 may be a mechanism for increasing the lifetime of the dimeric enzyme following dissociation from its activating complex at the cell membrane.     

DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 Polyubiquitination

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Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T_1/2 of 263 h, the mcRFB4s had T_1/2s ranging from 39–106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T_1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.Key words: chimeric, anti-CD22, monoclonal antibody, Fc mutations, ricin A chain, immunotoxins     

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.     

Genome assembly forensics: finding the elusive mis-assembly

We present the first collection of tools aimed at automated genome assembly validation. This work formalizes several mechanisms for detecting mis-assemblies, and describes their implementation in our automated validation pipeline, called amosvalidate. We demonstrate the application of our pipeline in both bacterial and eukaryotic genome assemblies, and highlight several assembly errors in both draft and finished genomes. The software described is compatible with common assembly formats and is released, open-source, at .     

Figaro: a novel statistical method for vector sequence removal

MOTIVATION: Sequences produced by automated Sanger sequencing machines frequently contain fragments of the cloning vector on their ends. Software tools currently available for identifying and removing the vector sequence require knowledge of the vector sequence, specific splice sites and any adapter sequences used in the experiment-information often omitted from public databases. Furthermore, the clipping coordinates themselves are missing or incorrectly reported. As an example, within the approximately 1.24 billion shotgun sequences deposited in the NCBI Trace Archive, as many as approximately 735 million (approximately 60%) lack vector clipping information. Correct clipping information is essential to scientists attempting to validate, improve and even finish the increasingly large number of genomes released at a 'draft' quality level. RESULTS: We present here Figaro, a novel software tool for identifying and removing the vector from raw sequence data without prior knowledge of the vector sequence. The vector sequence is automatically inferred by analyzing the frequency of occurrence of short oligo-nucleotides using Poisson statistics. We show that Figaro achieves 99.98% sensitivity when tested on approximately 1.5 million shotgun reads from Drosophila pseudoobscura. We further explore the impact of accurate vector trimming on the quality of whole-genome assemblies by re-assembling two bacterial genomes from shotgun sequences deposited in the Trace Archive. Designed as a module in large computational pipelines, Figaro is fast, lightweight and flexible. AVAILABILITY: Figaro is released under an open-source license through the AMOS package (http://amos.sourceforge.net/Figaro).     

Genome-Wide Analysis of Repetitive Elements in Papaya

Papaya (Carica papaya L.) is an important fruit crop cultivated in tropical and subtropical regions worldwide. A first draft of its genome sequence has been recently released. Together with Arabidopsis, rice, poplar, grapevine and other genomes in the pipeline, it represents a good opportunity to gain insight into the organization of plant genomes. Here we report a detailed analysis of repetitive elements in the papaya genome, including transposable elements (TEs), tandemly-arrayed sequences, and high copy number genes. These repetitive sequences account for ～56% of the papaya genome with TEs being the most abundant at 52%, tandem repeats at 1.3% and high copy number genes at 3%. Most common types of TEs are represented in the papaya genome with retrotransposons being the dominant class, accounting for 40% of the genome. The most prevalent retrotransposons are Ty3-gypsy (27.8%) and Ty1-copia (5.5%). Among the tandem repeats, microsatellites are the most abundant in number, but represent only 0.19% of the genome. Minisatellites and satellites are less abundant, but represent 0.68% and 0.43% of the genome, respectively, due to greater repeat length. Despite an overall smaller gene repertoire in papaya than many other angiosperms, a significant fraction of genes (>2%) are present in large gene families with copy number greater than 20. This repeat database clarified a major part of the papaya genome organization and partly explained the lower gene repertoire in papaya than in Arabidopsis.