The restriction endonuclease RflFII, isolated from Ruminococcus flavefaciens FD-1, recognizes the sequence 5''-AGTACT-3'', and is inhibited by site-specific adenine methylation

Abstract Citrate synthases from both thermophilic and halophilic Archaea have been purified to homogeneity using affinity chromatography on Matrex Gel Red A and elution with a combination of substrate (oxaloacetate) and product (coenzyme A). In a number of cases, purification from cell-extract to protein suitable for N-terminal sequencing can be achieved by this single-step procedure. The method is particularly useful in the rapid purification of a thermophilic archaeal citrate synthase from a cloned gene expressed in a mesophilic host.     

Isoniazid therapy of tuberculosis in baboons

A local epizootic of tuberculosis in a laboratory colony of baboons was controlled by 40–80 mg/kg daily dose of Isoniazid. There was definite regression of disease in all animals examined at autopsy, after three weeks and three months of therapy, following their use for unrelated acute experiments. One animal survives in apparent health with a healthy infant that was born after 102 days of therapy.     

Production and turnover ratio of Pisidium casertanum (Pelecypoda: Sphaeriidae) in the Ottawa River near Ottawa-Hull,Canada

Production of the sphaeriid clam Pisidium casertanum (Poli) was measured at several locations in the Ottawa River. Maximum production occurred in locations where sediment grain size was 3–4 phi units (0.250–0.0625 mm diameter, fine sand to very fine sand) and current velocity was near 0.1 m/sec. The turnover ratio of P. casertanum in the Ottawa River is a constant 3.8.     

Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase

Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Revisiting the historically important plant protein concanavalin A and reconstituting its biosynthesis in vitro reveals the advantages of its unusual biosynthesis via circular permutation.     

Surface properties are highly sensitive to small ph induced changes in the 3-D structure of alpha-lactalbumin

The change in structure of bovine alpha-lactalbumin in environments of decreasing pH from pH 7 to pH 3 was followed using high-resolution NMR and hydrogen exchange studies. The effect of the changes in the structure on the surface properties of the protein was also measured. As the pH was decreased from pH 7 toward pH 2, at which alpha-lactalbumin adopts a molten globule state, a small but increasing proportion of the molecules in the sample partially unfold. There was on average a loss of tertiary structure and a change in the environment of the tryptophan residues. A significant proportion of the change measured by both circular dichroism spectroscopy and interfacial methods observed as the pH was decreased from pH 7 to pH 4 was found to be irreversible upon readjustment back to pH 7. These changes in the sample conferred an increase in surface hydrophobicity and affected the surface properties. The surface activity was found to be highest at pH 4. This was because the increasing flexibility and surface hydrophobicity of the molecule with decreasing pH was balanced by the simultaneous increase in net charge repulsion. This conclusion was also confirmed by measurements of surface shear rheology. Interestingly the interfacial dilatational rheology was highest at the isoelectric point, indicating the dominant role of the charge interaction in controlling this parameter.     

Ecology of Uncultivated Oscillospira Species in the Rumen of Cattle, Sheep, and Reindeer as Assessed by Microscopy and Molecular Approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.