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Land and revenue in the Twelfth and Thirteenth Century: Normandy, England, the Holy Land Thanks to the documentation made available by the “Patrologia Latina” database, the author has managed to reconstruct the legal-institutional profile of the concessions ad firmam and their socio-economic contexts from the twelfth to the thirteenth centuries in Normandy, England, and the Holy Land. These agrarian contracts have usually escaped scholarly attention and are intriguing for their connection with the contractus censualis and the census reservativus, a legal institution present throughout the modern age and which played an important role in the revolutionaryn. M e d i t e r r a n e a R i c e r c h e s t o r i c h e Anno VII - Aprile 2010

Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araPBAD promoter oriented outward at one end (Tn5-araPBAD). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15°C) and in different media (rich and minimal medium). The Tn5-araPBAD-tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37°C) but not in the cold and in minimal medium. © 2004 Elsevier SAS. All rights reserved.

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site : BacTrx has an identity ranging from 45 to 53%with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl b-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 °C higher for BacTrx. The threedimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

The protein IF1 is a natural inhibitor of the mitochondrial FoF1-ATPase. Many investigators have been propted to identify the shortest segment of IF1, retaining its native activity, for use in biomedical applications. Here, the activity of the synthetic peptides IF1-(42-58) and IF1-(22-46) is correlated to their structure and conformational plasticity determined by CD and (1H)-NMR spectroscopy. Among all the IF1 segment tested, IF1-(42- 58) exerts the most potent, pH and temperature dependent activity on the FoF1 complex. The results suggest that, due to its flexible structure, it can fold in helical and/or b-spiral arrangements tha fovor the binding to the FoF1 complex, where tha native IF1 binds. IF1-(22-46), instead, as it adopts a rigid a-elical conformation, it inhibits ATP hydrolysis only in the soluble F1 mojety.

A total of 108 volunteers undergoing an elective surgical procedure were randomly given a single 2-g intravenous prophylactic dose of either a cephalosporin or mezlocillin. Stool samples were cultured for Clostridium difficile the day before the operation and later on postoperative days 4, 7, and 14. C. difficile was detected in 23.0% of patients who received a cephalosporin (cefoxitin, 8.3%; cefazolin, 14.3%; cefotetan, 20.0%; ceftriaxone, 25.0%; cefoperazone, 43.7%), in 3.3% of patients given mezlocillin, and in none of 15 control volunteers given no antimicrobial agent. No patient experienced diarrhea.