Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

Background  

Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein.     

The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.     

Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA

Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds.     

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.