1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution

Cytochrome b5 reconstituted with specifically deuterated hemins has led to the assignment of the resolved 6,7 beta-propionate protons and heme meso protons. Freshly reconstituted cytochrome b5 contains a mixture of two isomers in an approx. 1:1 ratio. As time proceeds the minor isomer decreases in intensity until the equilibrium ratio, approx. 8:1, of the two isomers is reached. The rate of the heme disorder kinetics was investigated for cytochrome b5 as a function of pH, oxidation state and 2,4 heme substitutents. Comparison of the kinetic data for cytochrome b5 with that obtained for other b-type heme proteins supports the proposal that the heme disorder arises from a 180 degree rotation of the heme about the alpha, gamma-meso axis. Computer-difference methods allow the spectra of the two individual isomers to be generated. Comparison of the NMR spectral parameters for the two individual isomers indicates small structural differences for amino acid side-chain orientations.     

Generation of chitin‐derived oligosaccharides toxic to pathogenic bacteria using ChiA74, an endochitinase native to Bacillus thuringiensis

Aims: To demonstrate that an endochitinase (ChiA74) native to Bacillus thuringiensis can be used to generate chitin‐derived oligosaccharides (OGS) with antibacterial activity against a number of aetiological agents of disease, including bacteria that cause diarrhoeal and emetic syndromes in humans. Methods and Results: The intact chiA74 with its cis elements was cloned into high and moderately high copy number Escherichia coli expression vectors. Functionally secreted ChiA74 was produced, and the endochitinase cleaved substrate colloidal chitin to produce OGS with 3, 5 and 6 degrees of polymerization. The enzyme was active for an extended period of incubation (24 h), but its activity showed a decrement of 73% and 87%, respectively, after 24 h of incubation at 37 and 55°C. OGS showed inhibitory activity against Bacillus cereus, Listeria inoccua, E. coli, Staphylococcus xylosus, Salmonella species, Staphylococcus aureus, Pseudomona aeruginosa, Shigella flexneri, and Proteus vulgaris. Conclusions: Endochitinase ChiA74 is able to stably maintain hydrolytic activity during prolonged incubation in a mix reaction with chitin to produce bioactive OGS with inhibitory activity against important food‐borne pathogenic bacteria. Significance and Impact of the Study: This is the first study showing that an endochitinase (ChiA74) native of the most important bioinsecticide used worldwide (B. thuringiensis), but here produced in E. coli, is able to generate chitin‐derived OGS with antibacterial activity against clinically significant food‐borne pathogenic bacteria.     

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients

Background  

Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein.     

Involvement of a plasmid in Escherichia coli envelope alterations.

A plasmid-containing wild-type Escherichia coli strain was treated with two plasmid-curing agents, sodium dodecyl sulfate and ethidium bromide. Plasmid elimination was accompanied by drastic changes in the morphology of the colonies. Analysis of the cured strain by scanning and transmission electron microscopy showed important alterations in size and morphology of the cells. Metabolic differences were also found between the wild-type and cured cells.