A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.     

Toward a comprehensive model for induced endoreduplication

Both the biological significance and the molecular mechanism of endoreduplication (END) have been debated for a long time by cytogeneticists and researchers into cell cycle enzymology and dynamics alike. Mainly due to the fact that a wide variety of agents have been reported as able to induce endoreduplication and the diversity of cell types where it has been described, until now no clear or unique mechanism of induction of this phenomenon, rare in animals but otherwise quite common in plants, has been proposed. DNA topoisomerase II (topo II), plays a major role in mitotic chromosome segregation after DNA replication. The classical topo II poisons act by stabilizing the enzyme in the so-called cleavable complex and result in DNA damage as well as END, while the true catalytic inhibitors, which are not cleavable-complex-stabilizers, do induce END without concomitant DNA and chromosome damage. Taking into account these observations on the induction of END by drugs that interfere with topo II, together with our recently obtained evidence that the nature of DNA plays an important role for chromosome segregation [Cortes, F., Pastor, N., Mateos, S., Dominguez, I., 2003. The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes. DNA Repair 2, 719-726.], a straightforward model is proposed in which the different mechanisms leading to induced END are considered.     

Multiplex analysis with peptide-encoded beads

Paramagnetic beads have considerable potential as identification tags in biological analysis. For example, magnetic sensor-based arrays using the magnetic field generated by paramagnetic beads to test hybridization between interacting molecules have attracted widespread interest in recent years. However, application of paramagnetic beads as identification tags is still limited, since they do not permit differentiation between samples for multiplex analysis. Here, we report the application of a novel encoding of paramagnetic beads with peptide sequences. This strategy allows DNA samples labeled with peptide-encoded paramagnetic beads to be identified by the selective enzymatic cleavage of each peptide cross-linker.     

Some factors affecting the stability of interferon alpha 2b in solution.

In this paper we evaluated the influence of the protein concentration and a formulation vehicle on the stability of recombinant human Interferon alpha 2b (rhIFN-alpha2b) in solution. The effect of the protein content (from 1 to 100 MIU/ml) at 37 degrees C, showed that higher concentration of this cytokine protected against the loss of bioactivity (antiviral titration) better than the lower concentrations. In contrast, rhIFN-alpha2b at 50 and 100 MIU/ml decreased the SDS/PAGE- and RP-HPLC-determined purity faster than samples at 1 or 10 MIU/ml. According to these results, 10 MIU/ml rhIFN-alpha2b was the best choice to evaluate the influence of a formulation on the stability of this cytokine. Taking this into consideration, we studied the stability of a liquid and albumin-free formulation of this protein at the recommended storage temperature (5+/-3 degrees C) and under accelerated conditions (28+/-2 degrees C). Accelerated storage results showed an acceptable biochemical stability of the active ingredient throughout 2 months. Real-time storage data confirmed the good biochemical stability of this formulation for 30 months.     

Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.     

The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis, and novel derivatives

Members of the aureolic acid family are tricyclic polyketides with antitumor activity which are produced by different streptomycete species. These members are glycosylated compounds with two oligosaccharide chains of variable sugar length. They interact with the DNA minor groove in high-GC-content regions in a nonintercalative way and with a requirement for magnesium ions. Mithramycin and chromomycins are the most representative members of the family, mithramycin being used as a chemotherapeutic agent for the treatment of several cancer diseases. For chromomycin and durhamycin A, antiviral activity has also been reported. The biosynthesis gene clusters for mithramycin and chromomycin A₃ have been studied in detail by gene sequencing, insertional inactivation, and gene expression. Most of the biosynthetic intermediates in these pathways have been isolated and characterized. Some of these compounds showed an increase in antitumor activity in comparison with the parent compounds. A common step in the biosynthesis of all members of the family is the formation of the tetracyclic intermediate premithramycinone. Further biosynthetic steps (glycosylation, methylations, acylations) proceed through tetracyclic intermediates which are finally converted into tricyclic compounds by the action of a monooxygenase, a key event for the biological activity. Heterologous expression of biosynthetic genes from other aromatic polyketide pathways in the mithramycin producer (or some mutants) led to the isolation of novel hybrid compounds.Felipe Lombó and Nuria Menéndez have equally contribute to this work.     

Computational disease gene identification: a concert of methods prioritizes type 2 diabetes and obesity candidate genes

Genome-wide experimental methods to identify disease genes, such as linkage analysis and association studies, generate increasingly large candidate gene sets for which comprehensive empirical analysis is impractical. Computational methods employ data from a variety of sources to identify the most likely candidate disease genes from these gene sets. Here, we review seven independent computational disease gene prioritization methods, and then apply them in concert to the analysis of 9556 positional candidate genes for type 2 diabetes (T2D) and the related trait obesity. We generate and analyse a list of nine primary candidate genes for T2D genes and five for obesity. Two genes, LPL and BCKDHA, are common to these two sets. We also present a set of secondary candidates for T2D (94 genes) and for obesity (116 genes) with 58 genes in common to both diseases.     

Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Rio Tinto (southwestern Spain)

The correlation between water physicochemical parameters and eukaryotic benthic composition was examined in Río Tinto. Principal component analysis showed a high inverse relationship between pH and most of the heavy metals analyzed as well as Dunaliella sp., while Chlamydomonas sp. abundance was positively related. Zn, Cu, and Ni clustered together and showed a strong inverse correlation with the diversity coefficient and most of the species analyzed. These eukaryotic communities seem to be more influenced by the presence of heavy metals than by the pH.     

Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Deltaklnde1 and Deltaklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum.     

Pyridazines. Part XXIX: synthesis and platelet aggregation inhibition activity of 5-substituted-6-phenyl-3(2H)-pyridazinones. Novel aspects of their biological actions

A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action.