HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.     

Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Protein kinase C modulates actin conformation in human T lymphocytes

We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes.     

Homodimeric and expanded behaviour of trimethylamine dehydrogenase in solution at different temperatures

Earlier studies using x-ray crystallography have shown that trimethylamine dehydrogenase (TMADH) from methylotropic bacteria exists as homodimers in the crystalline state. In this present hydrodynamic study we show that this is true also in dilute solution conditions and investigate the degree of swelling or relaxation of the protein in solution. Analytical ultracentrifugation was used to determine the molar mass and to investigate whether the homodimeric nature of this molecule in crystal form — as visualized by x-ray crystallography — is reproduced in dilute solution at temperatures between 4 and 40°C. The globular solution structure determined at 4 and 40°C is in good agreement with crystallographic data although trimethylamine dehydrogenase was found to be either more asymmetric in solution — or highly hydrated —, a phenomenon found to increase with temperature. In agreement with the crystallographic structure, the enzyme sediments as a homodimer with a molar mass of (163,000±5,000) g/mol. The concentration dependence of the sedimentation coefficient in the range of 0–1 mg/ml, indicates that no association or dissociation occurs. These findings are additionally supported by sedimentation equilibrium data in the concentration range of 0 to 1.8 mg/ml. Finally, from the sedimentation coefficient distribution at various temperatures, it was concluded that the enzyme is conformationally flexible and assumes an even more expanded structure at higher temperatures which is in good agreement with the hydrodynamic calculations performed. Correspondence to: S. E. Harding     

Mitigating the Impact of Bats in Historic Churches: The Response of Natterer’s Bats Myotis nattereri to Artificial Roosts and Deterrence

Bats frequently roost in historic churches, and these colonies are of considerable conservation value. Inside churches, bat droppings and urine can cause damage to the historic fabric of the building and to items of cultural significance. In extreme cases, large quantities of droppings can restrict the use of a church for worship and/or other community functions. In the United Kingdom, bats and their roosts are protected by law, and striking a balance between conserving the natural and cultural heritage can be a significant challenge. We investigated mitigation strategies that could be employed in churches and other historic buildings to alleviate problems caused by bats without adversely affecting their welfare or conservation status. We used a combination of artificial roost provision and deterrence at churches in Norfolk, England, where significant maternity colonies of Natterer’s bats Myotis nattereri damage church features. Radio-tracking data and population modelling showed that excluding M. nattereri from churches is likely to have a negative impact on their welfare and conservation status, but that judicious use of deterrents, especially high intensity ultrasound, can mitigate problems caused by bats. We show that deterrence can be used to move bats humanely from specific roosting sites within a church and limit the spread of droppings and urine so that problems to congregations and damage to cultural heritage can be much reduced. In addition, construction of bespoke roost spaces within churches can allow bats to continue to roost within the fabric of the building without flying in the church interior. We highlight that deterrence has the potential to cause serious harm to M. nattereri populations if not used judiciously, and so the effects of deterrents will need careful monitoring, and their use needs strict regulation.     

Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.