Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation.

A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines. The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues. The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances. The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29, 1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox. A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified. It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data. The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies. A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)     

The productive traits,fatty acid profile and nutritional indices of three lupin (Lupinus spp.) species cultivated in a Mediterranean environment for the livestock

The aim of this study was to investigate the productive traits and fatty acid profiles of eight sweet varieties of Lupinus albus, Lupinus angustifolius and Lupinus luteus cultivated in a Mediterranean environment (Sicily, southern Italy). This is being done to identify the most suitable species, from an agronomical and nutritional point of view, for supporting animal husbandry in this area. Seed yield, oil quantity and oil quality were considerably different depending on the genotype. L. albus showed the highest yield (2.0–2.4 t/ha), 100 seed weight (27.0 g, on average), oil content (70.4–75.5 g/kg), and n?3/n?6 polyunsaturated fatty acid ratio (0.39–0.51), L. angustifolius showed the highest content of saturated fatty acids (21.03–26.66 g/100 g) and L. luteus showed the highest levels of n?6 polyunsaturated fatty acids (47.56–50.85 g/100 g), unsaturated/saturated (4.93–5.62) and polyunsaturated/saturated (3.33–3.99) fatty acid ratios and the best atherogenic (0.06) and thrombogenic (0.09–0.11) indices. The data obtained suggests L. albus as the most interesting lupin species for aiding the crop-livestock food chain and L. luteus as a promising crop due to its high nutritive traits for the Mediterranean environment.     

Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e₁ bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e₁ bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.     

Fibrin Sealants in Dura Sealing: A Systematic Literature Review

BackgroundFibrin sealants are widely used in neurosurgery to seal the suture line, provide watertight closure, and prevent cerebrospinal fluid leaks. The aim of this systematic review is to summarize the current efficacy and safety literature of fibrin sealants in dura sealing and the prevention/treatment of cerebrospinal fluid leaks.MethodsA comprehensive electronic literature search was run in the following databases: Cochrane Database of Systematic Reviews, Cochrane Central Resister of Controlled Trials, clinicaltrials.gov, MEDLINE/PubMed, and EMBASE. Titles and abstracts of potential articles of interest were reviewed independently by 3 of the authors.ResultsA total of 1006 database records and additional records were identified. After screening for duplicates and relevance, a total of 78 articles were assessed by the investigators for eligibility. Thirty-eight were excluded and the full-text of 40 articles were included in the qualitative synthesis. Seven of these included only safety data and were included in the safety assessment. The remaining 33 articles included findings from 32 studies that enrolled a total of 2935 patients who were exposed to fibrin sealant. Among these 33 studies there were only 3 randomized controlled trials, with the remaining being prospective cohort analysis, case controlled studies, prospective or retrospective case series. One randomized controlled trial, with 89 patients exposed to fibrin sealant, found a greater rate of intraoperative watertight dura closure in the fibrin sealant group than the control group (92.1% versus 38.0%, p<0.001); however, post-operative cerebrospinal fluid leakage occurred in more fibrin sealant than control patients (6.7% versus 2.0%, p>0.05). Other clinical trials evaluated the effect of fibrin sealant in the postoperative prevention of cerebrospinal fluid leaks. These were generally lower level evidence studies (ie, not prospective, randomized, controlled trials) that were not designed or powered to demonstrate a significant advantage to fibrin sealant use. Two small case series studies evaluated the effect of fibrin sealants in persistent cerebrospinal fluid leak treatment, but did not establish firm efficacy conclusions. Specific adverse reports where fibrin sealants were used for dura sealing were limited, with only 8 cases reported in neurosurgical procedures since 1987 and most reporting only a speculative relationship/association with fibrin sealant exposure.ConclusionsA major finding of this systematic literature review is that there is a paucity of randomized studies that have evaluated the effectiveness and safety of fibrin sealants in providing intraoperative watertight dura closure and post-operative cerebrospinal fluid leakage. Among the limited studies available, evidence from a single randomized, controlled trial indicates that fibrin sealants provide a higher rate of intraoperative watertight closure of the dura suture line than control, albeit with a higher rate of postoperative cerebrospinal fluid leakage. Evidence from non-randomized, controlled trials suggests that fibrin sealants may be effective in preventing cerebrospinal fluid leaks with an acceptable safety profile. There is a substantial need for randomized, controlled clinical trials or well-designed prospective observational trials where the conduct of a randomized trial is not feasible to fully assess the impact of fibrin sealant utilization on the rates of intraoperative dura closure, postoperative cerebrospinal leakage, and safety.     

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Abstract

Tetracycline‐controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon‐γ in the RK‐13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon‐γ (rGPoIFN‐γ) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN‐γ cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN‐γ (up to 16 μg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of ³⁵S‐Methionine, labelled rGPoIFN‐γ with natural leukocytic IFN‐γ after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN‐γ species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N‐glycosydase F. The biological activity of rGPoIFN‐γ was in the same range as that of natural leukocytic PoIFN‐γ (2 × 10⁶ U/mg). Eventually, this recombinant mammalian IFN‐γ should constitute a very useful substitute for leukocyte PoIFN‐γ in in vitro or in vivo experiments.     

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.     

Comparison between paper and electronic sleep diary

This study aimed to test the concurrent validity of an electronic version (to run on tablet) of a sleep diary derived from the core Consensus Sleep Diary compared with the traditional paper and pencil version. To this end, 15 healthy volunteers (6 males; mean age 37.20 ± 17.55 years) every morning, for at least 7 consecutive days, filled both paper and electronic sleep diary. Furthermore, sleep was objectively assessed through actigraphy. With reference to all sleep parameters examined, no significant differences were observed between paper and electronic sleep diary. As expected, paper and electronic sleep diary showed the same poor agreement with actigraphic estimates of sleep quantity and sleep quality. On the basis of the present data, we can conclude that electronic sleep diary performs like paper sleep diary. Bearing in mind that electronic sleep diary presents several benefits in comparison to the paper sleep diary (e.g. avoiding of the “parking lot syndrome”, in which patient retrospectively completes more days at the same time; reducing the time for data entry and scoring), we suggest that, if the present findings will be confirmed also in clinical populations, electronic sleep diary should replace paper sleep diary in both research and clinical settings.