Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.     

Crystal structure of aspartyl dipeptidase from Xenopus laevis revealed ligand binding induced loop ordering and catalytic triad assembly

Gene encoding aspartyl dipeptidase from Xenopus levies (PepExl) is upregulated by thyroid hormone and is proposed to play a significant role in resorption of tadpole tail during metamorphosis. However, the importance of peptidase activity for the resorption of the tail remain elusive. Here we report the crystal structures of first eukaryotic S51 peptidase, PepExl, in its ligand-free and Asp-bound states at 1.4 and 1.8 Å resolutions, respectively. The active site is located at dimeric interface and the catalytic triad is found to be dissembled in ligand-free and assembled in Asp-bound state. Structural comparison and molecular dynamic simulations of ligand-free and Asp-bound states shows that distinct loop (loop-A) plays an important role in active site shielding, substrate binding and enzyme activation. This study illuminates the Asp-X dipeptide binding in PepExl is associated with ordering of the loop-A and assembly of residues of catalytic triad in active conformation for enzymatic activity.     

Of mice and the ‘Age of Discovery’: the complex history of colonization of the Azorean archipelago by the house mouse (Mus musculus) as revealed by mitochondrial DNA variation

Humans have introduced many species onto remote oceanic islands. The house mouse (Mus musculus) is a human commensal and has consequently been transported to oceanic islands around the globe as an accidental stowaway. The history of these introductions can tell us not only about the mice themselves but also about the people that transported them. Following a phylogeographic approach, we used mitochondrial D‐loop sequence variation (within an 849‐ to 864‐bp fragment) to study house mouse colonization of the Azores. A total of 239 sequences were obtained from all nine islands, and interpretation was helped by previously published Iberian sequences and 66 newly generated Spanish sequences. A Bayesian analysis revealed presence in the Azores of most of the D‐loop clades previously described in the domesticus subspecies of the house mouse, suggesting a complex colonization history of the archipelago as a whole from multiple geographical origins, but much less heterogeneity (often single colonization?) within islands. The expected historical link with mainland Portugal was reflected in the pattern of D‐loop variation of some of the islands but not all. A more unexpected association with a distant North European source area was also detected in three islands, possibly reflecting human contact with the Azores prior to the 15th century discovery by Portuguese mariners. Widening the scope to colonization of the Macaronesian islands as a whole, human linkages between the Azores, Madeira, the Canaries, Portugal and Spain were revealed through the sharing of mouse sequences between these areas. From these and other data, we suggest mouse studies may help resolve historical uncertainties relating to the ‘Age of Discovery’.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

Long‐term cytological and histological outcomes in women managed with loop excision treatment under local anaesthetic for high‐grade cervical intraepithelial neoplasia

Y. L. Woo, C. Badley, E. Jackson and R. Crawford Long‐term cytological and histological outcomes in women managed with loop excision treatment under local anaesthetic for high‐grade cervical intraepithelial neoplasia Objective: This study examines the impact of excision margin status after large loop excision of the transformation zone (LLETZ) under local anaesthetic for high‐grade cervical intraepithelial neoplasia (HG‐CIN) on the cytological and histological outcomes up to 5 years after treatment. Methods: Prospective cytological and histological data were obtained by examination of the colposcopy database at Addenbrooke’s Hospital, Cambridge, UK. All women aged between 19 and 50 years who underwent treatment for HG‐CIN by LLETZ under local anaesthetic were included in the study. Patients without follow‐up data were excluded from the study. The excision margin status was correlated with the subsequent cytological and histological outcomes. Results: A series of 967 women with CIN2 and CIN3 underwent LLETZ excision under local anaesthetic. Overall, 42% of women had disease present at the excision margin following LLETZ. Women with CIN3 were more likely than those with CIN2 to have an involved excision margin (P < 0.0001). Cytological recurrence was highest at 12 months (16%) and did not correlate with the CIN grade or excision margin status. Histological recurrence/persistence was also highest at 12 months follow‐up (15%) and this correlated with grade of CIN and margin status (P < 0.0001). Conclusions: Histological recurrence/persistence correlates with grade of CIN and excision margin status. Management of HG‐CIN in an outpatient setting under local anaesthetic is safe, cost effective and yields a favourable long‐term outcome.     

Influence of membrane fouling reducers (MFRs) on filterability of disperse mixed liquor of jet loop bioreactors

The effects of membrane fouling reducers (MFRs) (the cationic polyelectrolyte (CPE) and FeCI₃) on membrane fouling were studied in a lab-scale jet loop submerged membrane bioreactor (JL-SMBR) system. The optimum dosages of MFRs (CPE dosage = 20 mg g⁻¹MLSS, FeCI₃ dosage = 14 mg g⁻¹MLSS) were continuously fed to JL-SMBR system. The soluble and bound EPS concentrations as well as MLSS concentration in the mixed liquor of JL-SMBR were not changed substantially by the addition of MFRs. However, significant differences were observed in particle size and relative hydrophobicity. Filtration tests were performed by using different membrane types (polycarbonate (PC) and nitrocellulose mixed ester (ME)) and various pore sizes (0.45-0.22-0.1 μm). The steady state fluxes (J_ss) of membranes increased at all membranes after MFRs addition to JL-SMBR. The filtration results showed that MFRs addition was an effective approach in terms of improvement in filtration performance for both membrane types.