The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.     

Effect of maternal dietary exclusion on breast fed infants with eczema: two controlled studies.

Thirty seven breast fed infants with eczema were studied to see whether changes in their mothers'' diets affected their skin condition. Nineteen mothers and babies took part in a double blind crossover trial of exclusion of egg and cows'' milk, and 18 took part in open exclusion of 11 foods followed by double blind challenge to those mothers whose infants seemed to respond. Babies were examined at the beginning and end of each dietary period, and the extent and severity of the rash were given a numerical score. The eczema improved in six infants when their mothers avoided egg and cows'' milk and worsened again when these were reintroduced. Two infants suffered gastrointestinal reactions after maternal ingestion of egg and cows'' milk, one developing colitis. Maternal dietary exclusion seems to benefit some breast fed babies with eczema.     

Occurrence of toxigenic Clostridium botulinum type C in the soil of wetlands in Saskatchewan

Mouse-lethal toxin identified as that of Clostridium botulinum type C by antitoxin neutralization was present in cultures of 38.0% of 326 soil samples collected from 28 wetlands in Saskatchewan. There was no difference in prevalence of toxicity between samples collected in spring and summer, and no relationship was evident between the occurrence of toxicity and water salinity, marsh type or water depth. There was a strong association between the prior occurrence of avian botulism in a marsh and the presence of toxin in cultures from soil; 59.2% of soil samples from marshes with a known history of botulism produced toxin, whereas only 6.2% of soil samples from marshes with no history of the disease produced toxin. Eight of the 10 soil samples collected from a marsh that had been dry for several years, and from another marsh that had not had a recognized outbreak of botulism for 11 yr produced toxin, indicating a long residual effect after a botulism outbreak. The results suggest that any wetland with a history of botulism is likely to suffer repeated occurrences because of heavy contamination of the soil with spores, and should be managed to control the disease.     

Trade and habitat change virtually eliminate the Grey Parrot Psittacus erithacus from Ghana

The heavily traded Grey Parrot Psittacus erithacus is believed to have undergone rapid population decline, yet there are almost no quantitative data on abundance changes over time from anywhere within its huge range. We reviewed the species’ historical abundance across Ghana, undertook targeted searches during 3‐ to 5‐day visits to 42 100‐km² cells across the country's forest zone, repeated counts at 22 parrot roosts first performed two decades ago and gauged around 900 people's perceptions of the decline and its causes. In over 150 days of fieldwork, just 32 groups (maximum group size = 12) were recorded in 10 cells. Encounter rates averaged 0.15 individuals per hour of targeted search, around 15 times lower than those recorded in the early 1990s. No active roosts were found, and only 18 individuals were recorded in three roost areas that each harboured 700–1200 birds two decades ago. Interviewees stressed the importance of very tall trees of commercially important species such as Terminalia superba and Ceiba pentandra for nesting and roosting, and believed that the felling of large trees on farmland (42% of responses) and trapping for trade (37%) were the two main causes of decline. Ghana has lost 90–99% of its Grey Parrots since 1992, a time when the population had presumably already been seriously reduced by two decades of extremely heavy trade. There is no evidence that, away from one or two localities, declines are less severe anywhere else within the West African range of P. erithacus, or across the entire range of the recently split Timneh Parrot Psittacus timneh.     

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2–3?kcal?mol^?1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel.     

Effects of preparation method, age, and plating technique of thin agar layer media on recovery of Escherichia coli O157:H7 injured by sodium chloride

The thin agar layer (TAL) method was experimentally tested to determine its ability to recover Escherichia coli O157:H7 injured by sodium chloride (NaCl). Cells grown in Brain Heart Infusion broth with 0%, 5%, or 7.5% (w/v) NaCl were spread and spiral plated onto Tryptic Soy agar (TSA), MacConkey Sorbitol agar (MSA), and TSA/MSA TAL combinations. Generally, TSA recovered more injured cells than TAL (p < or =0.05), and TAL recovered more cells than MSA (p < or =0.05). Preparation mode (two vs. three layers) and age (0, 1, or 7 days) of TAL had negligible effect on resuscitation of injured cells (p > 0.05). TAL, which is conventionally used to recover heat, cold, and acid-injured foodborne pathogens, may be used to recover NaCl-injured E. coli O157:H7.     

Exploiting protein structure data to explore the evolution of protein function and biological complexity

New directions in biology are being driven by the complete sequencing of genomes, which has given us the protein repertoires of diverse organisms from all kingdoms of life. In tandem with this accumulation of sequence data, worldwide structural genomics initiatives, advanced by the development of improved technologies in X-ray crystallography and NMR, are expanding our knowledge of structural families and increasing our fold libraries. Methods for detecting remote sequence similarities have also been made more sensitive and this means that we can map domains from these structural families onto genome sequences to understand how these families are distributed throughout the genomes and reveal how they might influence the functional repertoires and biological complexities of the organisms. We have used robust protocols to assign sequences from completed genomes to domain structures in the CATH database, allowing up to 60% of domain sequences in these genomes, depending on the organism, to be assigned to a domain family of known structure. Analysis of the distribution of these families throughout bacterial genomes identified more than 300 universal families, some of which had expanded significantly in proportion to genome size. These highly expanded families are primarily involved in metabolism and regulation and appear to make major contributions to the functional repertoire and complexity of bacterial organisms. When comparisons are made across all kingdoms of life, we find a smaller set of universal domain families (approx. 140), of which families involved in protein biosynthesis are the largest conserved component. Analysis of the behaviour of other families reveals that some (e.g. those involved in metabolism, regulation) have remained highly innovative during evolution, making it harder to trace their evolutionary ancestry. Structural analyses of metabolic families provide some insights into the mechanisms of functional innovation, which include changes in domain partnerships and significant structural embellishments leading to modulation of active sites and protein interactions.