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Informational content of the echinoderm egg.

The sea urchin egg contains a store of mRNA synthesized during oogenesis but translated only after fertilization, which accounts for a large, rapid increase in the rate of synthesis of largely the same set of proteins synthesized by eggs. Starfish oocytes contain a population of stored maternal mRNA that becomes actively translated upon GVBD and codes for a set of proteins distinct from that synthesized by oocytes. The sequence complexity of RNA in echinoderm eggs is about 3.5 x 10(8) nucleotides, enough to code for about 12,000 different mRNAs averaging 3 kb in length. About 2-4% of the egg RNA functions as mRNA during early embryonic development; most of the sequences are rare, represented in a few thousand copies per egg, but some are considerably more abundant. Many of the stored RNA sequences accumulate during the period of vitellogenesis, which lasts a few weeks. The mechanisms of storage and translational activation of maternal mRNA are not well understood. Histone mRNAs are sequested in the egg pronucleus until first cleavage, but other mRNAs are widely distributed in the cytoplasm. The population of maternal RNA includes many very large molecules having interspersed repetitive sequence transcripts colinear with single-copy sequences. The structural features of much of the cytoplasmic maternal RNA is thus reminiscent of incompletely processed nuclear precursors of mRNA. The functional role of these strange molecules is not understood, but many interesting possibilities have been considered. For instance, they may be segregated into different cell lineages during cleavage and/or they may become translationally activated by selective processing during development. Maternal mRNA appears to be underloaded with ribosomes when translated, possibly because the coding sequences are short relative to the size of the mRNA. Most abundant and many rare mRNA sequences persist during embryonic development. The rare sequence molecules are replaced by newly synthesized RNA, but some abundant maternal transcripts appear to persist throughout embryonic development. Most of the proteins present in the egg do not change significantly in mass during development, but a few decline or accumulate substantially. Together, these observations indicate that much of the information for embryogenesis is stored in the egg, although substantial changes in gene expression occur during development.

Animals↗

Electron microscopic study of human immunodeficiency virus type 1 (HIV-1) core structure: two RNA strands in the core of mature and budding particles.

The basic structure of human immunodeficiency virus type 1 (HIV-1) has been investigated morphologically; however, the internal structure of HIV-1 core is not well understood. We studied the internal structures by transmission electron microscopy. We modified the method for electron staining of ultrathin sections and processed electron microscopic photographs using a computer. We confirmed that a mature HIV-1 particle had two copies of RNA strands in a cone-shaped core. These two RNA strands formed a coiling structure and interwound each other, and were already present in the late budding stage.

HIV-1↗

Self-cleaving motifs are found in close proximity to the sites utilized for U16 snoRNA processing.

A class of small nucleolar RNAs (snoRNAs) is encoded in introns of protein-coding genes. The U16 snoRNA belongs to this class; it is encoded in the third intron of the Xenopus laevis (Xl) L1 ribosomal protein encoding gene and is released from the pre-mRNA by processing both in vivo and in vitro systems. In this paper, we show that in close proximity to the U16 snoRNA processing sites, sequences displaying self-cleaving activity are present. These elements are conserved in the two copies of the Xl L1 and in the single copy of the X. tropicalis L1. The catalytic activity corresponds to that already described for the minimal hairpin ribozyme [Dange et al., Science 242 (1990) 585-588]; it is Mn(2+)-dependent, produces 2'-3' cyclic phosphate and 5'-OH termini and comprises an essential GAAA element. Here we show that the 2'-OH group of the G residue is essential for catalysis.

Animals↗

Natural and experimental prion diseases of humans and animals.

Prions cause transmissible and genetic neurodegenerative diseases. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc), which is encoded by a chromosomal gene. Although the PrP gene is single copy, transgenic mice with both alleles of the PrP gene ablated develop normally. A post-translational process, as yet unidentified, converts the cellular prion protein (PrPC) into PrPSc. Scrapie incubation times, neuropathology and prion synthesis in transgenic mice are controlled by the PrP gene. Mutations in this gene are genetically linked to the development of neurodegeneration. Transgenic mice expressing mutant PrP spontaneously develop neurological dysfunction and spongiform neuropathology. Future investigations of prion diseases using molecular biological and genetic approaches promise to yield much new information about these once enigmatic disorders.

Animals↗

2-micron circle plasmids do not reduce yeast life span.

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir+ and cir0 strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.

Particle Size↗

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina.

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.

5' Untranslated Regions↗

The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris.

The methylotrophic yeast Pichia pastoris is a popular host for the production of a variety of recombinant proteins. We describe the use of a novel selectable marker, the P. pastoris formaldehyde dehydrogenase gene (FLD1) for DNA-mediated transformations of this yeast. The product of the FLD1 gene (Fld1p) is required for growth of P. pastoris on methanol as a carbon source and methylamine as a nitrogen source. In both these C(1) pathways, Fld1p oxidizes formaldehyde to formate, which is subsequently further oxidized by a second dehydrogenase to carbon dioxide. We show that the FLD1 gene can be used as a marker in transformations of a P. pastoris fld1 host by selection on plates containing methylamine. Furthermore, we demonstrate that populations of these transformants can be enriched for strains that receive multiple copies of an FLD1-based vector by their increased resistance to formaldehyde. We provide the FLD1 selection system in a set of P. pastoris expression vectors that are composed almost entirely of P. pastoris DNA (except for the recombinant gene) and are devoid of antibiotic resistance genes or other sequences of bacterial origin. The vectors are useful for the selection of strains containing multiple copies of an expression vector and may be ideal for certain large-scale recombinant protein production processes where strains containing non-P. pastoris DNA sequences, particularly bacterial antibiotic resistance genes and replication origins, are considered a potential biological hazard to be avoided.

Aldehyde Oxidoreductases↗

A data-gathering broker as a future-orientated approach to supporting EPR users.

With the continued expansion of electronic patient record systems ahead of comprehensive evidence, metrics, or future-proofing, health informatics in Europe and beyond is embarking on a faith-driven adventure that also risks data swamping of end-users. An alternative approach is an information broker system, drawing from departmental data sources. A 3-year study in health and social care has produced a first demonstrator which can search for specified information in heterogeneous distributed data stores, with source-specific permission can copy it, and then merge the search results into one integrated picture in a real-time process which is also captured in an audit system. The research project has addressed a number of issues during the study, including updating the concepts of role-based access, semantic interoperability, and harnessing web-based services bound at the time of need. A demonstrator now exists, and provides a platform for further application and development research. This paper summarises how this opens up a viable alternative approach for the next generation of health record systems, enabling record searching and integration as and when it is needed for specific patient-related purposes, whilst being independent of organisations, diagnostic approaches, or service delivery structures, and reducing the risks of data swamping.

Access to Information↗

Identifying corollary discharges for movement in the primate brain.

The brain keeps track of the movements it makes so as to process sensory input accurately and coordinate complex movements gracefully. In this chapter we review the brain's strategies for keeping track of fast, saccadic eye movements. One way it does this is by monitoring copies of saccadic motor commands, or corollary discharges. It has been difficult to identify corollary discharge signals in the primate brain, although in some studies the influence of corollary discharge, for example on visual processing, has been found. We propose four criteria for identifying corollary discharge signals in primate brain based on our experiences studying a pathway from superior colliculus, in the brainstem, through mediodorsal thalamus to frontal eye field, in the prefrontal cortex. First, the signals must originate from a brain structure involved in generating movements. Second, they must begin just prior to movements and represent spatial attributes of the movements. Third, eliminating the signals should not impair movements in simple tasks not requiring corollary discharge. Fourth, eliminating the signals should, however, disrupt movements in tasks that require corollary discharge, such as a double-step task in which the monkey must keep track of one saccade in order to correctly generate another. Applying these criteria to the pathway from superior colliculus to frontal eye field, we concluded that it does indeed convey corollary discharge signals. The extent to which cerebral cortex actually uses these signals, particularly in the realm of sensory perception, remains unknown pending further studies. Moreover, many other ascending pathways from brainstem to cortex remain to be explored in behaving monkeys, and some of these, too, may carry corollary discharge signals.

Animals↗

Regulation of Golgi structure through heterotrimeric G proteins.

We have previously shown that ilimaquinone (IQ), a marine sponge metabolite, causes complete vesiculation of the Golgi stacks. By reconstituting the IQ-mediated vesiculation of the Golgi apparatus in permeabilized cells, we now demonstrate that this process does not require ARF and coatomers, which are necessary for the formation of Golgi-derived COPI vesicles. We find that IQ-mediated Golgi vesiculation is inhibited by G alpha(s)-GDP and G alpha(i3)-GDP. Interestingly, adding betagamma subunits in the absence of IQ is sufficient to vesiculate Golgi stacks. Our findings reveal that IQ-mediated Golgi vesiculation occurs through activation of heterotrimeric G proteins and that it is the free betagamma, and not the activated alpha subunit, that triggers Golgi vesiculation.

Animals↗

Babesia divergens: cloning and biochemical characterization of Bd37.

The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are described. In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

Amino Acid Sequence↗

Purification and biochemical characterization of a soluble alpha-glucosidase from the parasite Entamoeba histolytica.

A soluble alpha-glucosidase presumably involved in the general carbohydrate metabolism was purified from E. histolytica trophozoites by a three-step procedure consisting of ion exchange, size exclusion and adsorption chromatographies in columns of Mono Q, Sepharose CL-6B and hydroxyapatite, respectively. After the last step, the enzyme was enriched about 673-fold over the starting material with a yield of 18%. SDS-PAGE revealed the presence in the purified preparations of two polypeptides of comparable intensity exhibiting molecular weights of 43 and 68 kDa. These results and the molecular weight of 243 kDa determined by gel filtration, suggest that the native enzyme is a heterotetramer consisting of two copies of each subunit. Some properties were investigated to determine the role of this activity in glycoprotein processing. Analysis of linkage specificity using a number of substrates indicated a preferential hydrolysis of isomaltose (alpha1,6) with much less activity on nigerose (alpha1,3) and maltose (alpha1,4). Trehalose (alpha1,1), kojibiose (alpha1,2) and cellobiose (beta1,4) were not cleaved at all. As expected, isomaltose competed away hydrolysis of 4-methylumbelliferyl-alpha-D-glucoside with a higher efficiency than nigerose and maltose. Hydrolysis of the fluorogenic substrate was competitively inhibited by glucose and 6-deoxy-D-glucose with comparable K(i) values of 0.23 and 0.22 mM, respectively. Sensitivity of the enzyme to the alpha-glucosidase inhibitors 1-deoxynojirimycin, castanospermine and australine largely depended on the substrate utilized to determine activity. 1-Deoxynojirimycin and castanospermine inhibited isomaltose hydrolysis in a competitive manner with K(i) values of 1.2 and 1.5 muM, respectively. The properties of the purified enzyme are consistent with a general glycosidase probably involved in glycogen metabolism.

Animals↗

Crystal structure of the von Willebrand factor A1 domain in complex with the function blocking NMC-4 Fab.

The presence of one or more copies of von Willebrand factor type A domains identifies a superfamily of proteins usually involved in biological processes controlled by specific molecular interactions, often adhesive in nature. We have solved the crystal structure of the prototypic von Willebrand factor A1 domain, essential for the antihemorrhagic activity of platelets, in complex with the function blocking antibody, NMC-4, at 2.2 A resolution. This has led to the recognition of a putative binding groove for the platelet receptor, glycoprotein Ib alpha, formed by two adjacent alpha-helices and a beta-strand. The structure also shows a contact interface between A1 domain pairs, suggesting a hypothetical mechanism for the regulation of protein assembly and heterologous ligand binding mediated by homophilic interactions of type A domains.

Amino Acid Sequence↗

Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain.

Haem-containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H2O2 detoxification. But, to date, no haem-containing enzymatic defence against toxic H2O2 has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant-like APX (ascorbate peroxidase) from Leishmania major (LmAPX) and characterized its catalytic parameters under steady-state conditions. Examination of its protein sequence indicated approx. 30-60% identity with other APXs. The N-terminal extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acids containing a transmembrane domain. To understand how the transmembrane domain influences the structure-function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate-binding affinity but slowed down ascorbate oxidation and increased resistance to H2O2-dependent inactivation in the absence of electron donor by 480-fold. Spectral studies show that H2O2 can quickly oxidize the native enzyme to compound (II), which subsequently is reduced back to the native enzyme by an electron donor. In contrast, ascorbate-free transmembrane domain-containing enzyme did not react with H2O2, as revealed by the absence of compound (II) formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H2O2 that is generated by endogenous processes and as a result of external influences such as the oxidative burst of infected host macrophages or during drug metabolism by Leishmania.

Amino Acid Sequence↗

The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates.

Previous work has shown that RNase E-mediated cleavage of RNAI, an antisense repressor of the replication of ColE1-type plasmids, relieves repression in vivo by endonucleolytically converting RNAI to a rapidly decaying product. We report that mutations in the Escherichia coli pcnB gene result in a 10-fold prolongation of the half-life of RNAI decay intermediates and also of truncated RNAI primary transcripts lacking sites attacked by RNase E. Using Northern blotting, primer extension analysis, [32P]GTP capping of 5'-triphosphate termini, and PCR amplification methods, we show that pcnB-mediated acceleration of RNAI degradation is associated with posttranscriptional 3' addition of adenosine residues in vivo to native and processed forms of RNAI. Accumulation of antisense RNAI decay products in pcnB mutants potentially explains the reduced copy number of ColE1-type plasmids seen in the mutated bacteria.

Adenosine Monophosphate↗

Biological cells as templates for hollow microcapsules.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Biotechnology↗

ArrayCyGHt: a web application for analysis and visualization of array-CGH data.

UNLABELLED: ArrayCyGHt is a web-based application tool for analysis and visualization of microarray-comparative genomic hybridization (array-CGH) data. Full process of array-CGH data analysis, from normalization of raw data to the final visualization of copy number gain or loss, can be straightforwardly achieved on this arrayCyGHt system without the use of any further software. ArrayCyGHt, therefore, provides an easy and fast tool for the analysis of copy number aberrations in any kinds of data format. AVAILABILITY: ArrayCyGHt can be accessed at http://genomics.catholic.ac.kr/arrayCGH/

Algorithms↗

Basal temporal language area.

Language interference was elicited by electrical stimulation of the dominant basal temporal region in 8 out of 22 cases and in none of 7 cases with subdural electrodes implanted over the nondominant temporal lobe. Language interference was elicited by stimulation of electrodes placed over the fusiform gyrus 3-7 cm from the tip of the temporal lobe. Electrical stimulation of the basal temporal language area produced a global receptive and expressive aphasia with speech arrest at high stimulus intensities. Other higher cortical function, for example copying complex designs or memory of nonverbal information was intact, in spite of the total inability to process verbal information. At lower stimulus intensities partial aphasias with a predominant receptive component occurred. Surgical resection of the basal temporal language area produces no lasting language deficit.

Adolescent↗