Use of screws and cement in revision TKA with primary or revision specific prosthesis with up to 17 years followup.

The purpose of this study was to investigate the use for screws and cement, and primary and revision specific prosthesis for revision TKA. Between July 1989 and February 2010, 839 consecutive revision TKAs were performed, with 609 knees meeting inclusion criteria. At 17 years followup, Kaplan-Meier survivorship was 0.9859 for revision specific prosthesis with screws and cement, 0.9848 for revision prosthesis with no screws, 0.9118 for primary prosthesis with screws, and 0.9424 for primary prosthesis with no screws. Revision TKAs using screws had greater defects (P<.0001). Use of revision prosthesis along with screws and cement to correct largely defective revision TKAs is highly recommended.

Use of screws and cement in primary TKA with up to 20 years follow-up.

The purpose of this study was to investigate screws and cement for large tibial bone defects during primary TKA. Of 14,686 consecutive primary TKAs performed between December 1988 and February 2010, 256 received screws and cement for tibial defects. Cox regression was used for the analysis. 20-year survival probability was 0.9897 (screws) and 0.9339 (no screws) (P = .4225 log-rank). Tibial bone condition was significantly worse in knees receiving screws (P < .0001) with 73.0% having defects in the screws group and 3.4% (P < .0001) for non-screws. Radiolucency appeared in 13.7% (screws) and 6.4% (no screws) postoperatively. Screws were $137 each, wedges $910 to $2240. Knees with tibial defects and screws performed similarly if not better than knees without defects at substantially lower cost than alternatives.

Author Correction: Single compounds elicit complex behavioural responses in wild, free-ranging rats.

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Single compounds elicit complex behavioural responses in wild, free-ranging rats.

There is mounting evidence that single compounds can act as signals and cues for mammals and that when presented at their optimal concentration they can elicit behavioural responses that replicate those recorded for complex mixtures like gland secretions and foods. We designed a rapid bioassay to present nine compounds that we had previously identified in foods, each at seven different concentrations (63 treatments), to wild, free-ranging rats and scored each treatment for attraction and three behavioural responses. Nine treatments (taken from five compounds) statistically outperformed the current standard rat attractant, peanut butter. Attraction to treatments was highest at the two lowest concentrations (0.1 and 0.01 µg g-1) and a statistically significant relationship of increasing attraction with decreasing treatment concentration was identified. Our study identified five compounds not previously associated with behavioural responses by rats that elicit equivalent or more intense behavioural responses than those obtained with peanut butter. Moreover, attraction to treatments was driven by a concentration-dependent relationship not previously reported. This is the first study to identify isopentanol, 1-hexanol, acetoin, isobutyl acetate and 2-methylbutyl acetate as possible semiochemicals/cues for rats. More broadly, our findings provide important guidance to researchers in the ongoing search for mammalian semiochemicals and cues.

Bioactivity-Guided Metabolite Profiling of Feijoa ( Acca sellowiana) Cultivars Identifies 4-Cyclopentene-1,3-dione as a Potent Antifungal Inhibitor of Chitin Synthesis.

Pathogenic fungi continue to develop resistance against current antifungal drugs. To explore the potential of agricultural waste products as a source of novel antifungal compounds, we obtained an unbiased GC-MS profile of 151 compounds from 16 commercial and experimental cultivars of feijoa peels. Multivariate analysis correlated 93% of the compound profiles with antifungal bioactivities. Of the 18 compounds that significantly correlated with antifungal activity, 5 had not previously been described from feijoa. Two novel cultivars were the most bioactive, and the compound 4-cyclopentene-1,3-dione, detected in these cultivars, was potently antifungal (IC50 = 1-2 µM) against human-pathogenic Candida species. Haploinsufficiency and fluorescence microscopy analyses determined that the synthesis of chitin, a fungal-cell-wall polysaccharide, was the target of 4-cyclopentene-1,3-dione. This fungal-specific mechanism was consistent with a 22-70-fold reduction in antibacterial activity. Overall, we identified the agricultural waste product of specific cultivars of feijoa peels as a source of potential high-value antifungal compounds.

ACL Damage and Deficiency is Associated with More Severe Preoperative Deformity, Lower Range of Motion at the Time of TKA.

BACKGROUND: ACL status varies in the arthritic knee during TKA. QUESTIONS/PURPOSES: The purpose of this study was to examine clinical features and intraoperative findings associated with stages of ACL degeneration. METHODS: Coronal deformity, ROM, intra-articular degenerative patterns, and ligament releases were assessed for 1656 knees during TKA. Common patterns of deformity and severity of degenerative change were assessed as a function of the severity of ACL deficiency. RESULTS: Of the 1656 knees assessed, 27% had a normal ACL, 55% exhibited damage, and 18% exhibited complete absence of the ACL. Increased coronal deformity and lower preoperative ROM was associated with ACL deficiency. Increased chondral and meniscal damage and more extensive osteophyte formation were also found. More extensive ligament releases were required in ACL-deficient knees. CONCLUSIONS: The status of the ACL is predictive of the need for increased surgical deformity correction. A better understanding of ACL status is an important consideration during in choosing TKA as opposed to unicompartmental arthroplasty. The status of the ACL should be considered in planning for implant choice in TKA.

Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases.

The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha.

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Brönsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.

Studies on the formation and incorporation of streptolidine in the biosynthesis of the peptidyl nucleoside antibiotic streptothricin F.

Streptothricin F (STF, 1) is a peptidyl nucleoside antibiotic produced by Streptomyces lavendulae. Studies were conducted to address the formation and timing of incorporation of the arginine-derived base streptolidine (4) during the biosynthesis of 1. [guanidino-(13)C]Streptolidine (10) was prepared by modification of an established method and used in whole-cell incorporation experiments. Analysis of the purified STF by (13)C NMR revealed a 1.9% enrichment of the guanidino carbon, confirming 4 as an advanced precursor to 1 and supporting proposals that 1 is assembled via a convergent biosynthetic pathway. To identify advanced intermediates in the conversion of L-arginine to 4, (2S,3R)-[guanidino-(13)C]capreomycidine (32) was prepared from oxazolidine aldehyde (18) via 1,1-dimethylethyl (4R,1'S)-4-(1',3'-diaminopropyl)-2,2-dimethyl-3-oxazolidinecarboxylate (30). Treatment of 30 with Br(13)CN yielded the corresponding diprotected amino alcohol, which was readily converted to 32. The STF isolated from whole-cell incorporation experiments with 32 showed no significant (13)C enrichment at the guanidino carbon. These results suggest that 32 may be an enzyme-bound intermediate, unable to enter the cell, or is not a precursor to STF.

Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation.

Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD(+) and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr). Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging. Here we examine the role of NAD(+) metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro. Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates. Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue. Both ends of the dinucleotide NAD(+) are shown to be critical for high selectivity and high affinity. Among the NAD(+) metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site. Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr. The presented findings also suggest that changes in the physiological NAD(+):NADH ratio, without a change in NAD(+), would yield little alteration in Sir2 activity. That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC(50) of 17 mm). We propose that changes in both free nicotinamide and free NAD(+) afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration.

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases.

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brönsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.

Probing the transition-state structure of dual-specificity protein phosphatases using a physiological substrate mimic.

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective timing of the proton transfer to the leaving group and P-O fission; with the alkyl ester substrate, protonation is ahead of P-O fission, while with the aryl substrate, the two processes are more synchronous. Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement of Asp-92 in protonating the ester oxygen, either in a step prior to significant P-O bond cleavage or in a concerted but asynchronous mechanism in which protonation is ahead of P-O bond fission. Collectively, the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of the proton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over with similar catalytic efficiency.

Conserved enzymatic production and biological effect of O-acetyl-ADP-ribose by silent information regulator 2-like NAD+-dependent deacetylases.

Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD(+)-dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.