Method of alkalization and monitoring of urinary pH for prevention of recurrent uric acid urolithiasis: a systematic review.

Uric acid (UA) urolithiasis comprises around 5-10% of all stones and can frequently recur. Due to the fact that UA stones form in acidic urine with a pH <5.5, these patients require special attention compared to other stone patients. The international guidelines suggest treatment and metaphylaxis by urinary alkalization. The objective of this review is to critically asses the available evidence concerning the method and efficacy of this treatment modality. A systematic review on the methods of metaphylactic therapy using oral alkalization of UA urolithiasis was conducted by two authors. Evidence was sought using a predefined search strategy in seven different databases. The provided evidence was critically evaluated using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Cochrane collaboration tool for assessing the risk of bias. Twelve manuscripts were included of which one was a randomised trial. They focussed on ways to alkalize urine and its effect on stone recurrence. Because of their methodology and heterogeneity, the evidence is presented in a narrative review. There were differences in medication used for alkalizing urine, ways of monitoring urine pH and evaluating its efficacy. The reported outcomes also differed between studies. There is currently a lack of clear evidence for the method of alkalization of urine and the method of pH measurement. Besides this, for an established treatment modality, there is lack of long term results for the alkalization therapy. In conclusion, urine alkalization is an established treatment modality for the metaphylaxis of UA urolithiasis despite the lack of evidence from high quality studies on the methods of alkalization and its treatment efficacy. The studies published on this topic are scarce and contain notable risks of bias which should be kept in mind when interpreting the stated results.

Laboratory evaluation of the Coasys® Plus C coagulation analyzer.

INTRODUCTION: The Coasys® Plus C (Behnk Elektronik, distributed by Roche Diagnostics) is a coagulation analyzer for small to midsize clinical chemistry laboratories. We performed a laboratory evaluation. MATERIALS AND METHODS: After a familiarization period the dead volume, carry-over, capacity, within-assay reproducibility and imprecision were determined for the tests aPTT (STA APTT en STA Cephascreen), PT (STA Neoplastin Plus, STA Neoplastin R), INR (Hepato Quick), fibrinogen (STA Fibrinogen), antithrombin (Antithrombin III) and D-Dimer (Tina-quant D-Dimer Gen.2). A precision test and patient comparison with a STA-R Evolution® system were performed for aPTT (STA Cephascreen), PT (STA Neoplastin R), fibrinogen, antithrombin and D-Dimer. RESULTS: No carry-over was detected. The analyzer performed on average 66 measurements per hour. Within-assay reproducibility (% with normal/abnormal/extreme result): STA APTT 2.4/1.7/2.7; aPTT Cephascreen 1.4/3.1/1.8; PT Neoplastin Plus 1.2/2.1/1.7; PT Neoplastin R 2.3/-/3.1; INR Hepato Quick 0.6/0.9/1.9; fibrinogen 4.8/3.0/4.5; antithrombin 1.3/4.1/3.8; D-Dimer 19.9/4.3/4.3. Total imprecision (% with control 1/control 2/human pooled plasma): STA APTT 3.1/1.4/2.1; APTT Cephascreen 2.3/2.6/2.0; PT Neoplastin Plus 3.3/1.3/4.1; PT Neoplastin R 3.7/4.0/3.5; INR Hepato Quick 1.5/4.3/1.4; fibrinogen 3.4/5.8/8.1; antithrombin 3.0/6.4/2.4; D-Dimer 4.6/2.2/30.3. The correlation between the Coasys® Plus C and the STA-R Evolution® was good for aPTT, PT, antithrombin and fibrinogen. Some differences were observed for extreme deviant results for fibrinogen. For the analysis of D-Dimer, no sufficient correlation was found between the two analyzers. CONCLUSIONS: The Coasys® Plus C analyzer is fast, easy to handle and safe for the personnel. Its analytical performance makes it suitable for use in a clinical chemistry laboratory.

RING1B ubiquitination and stability are regulated by ARF.

The activity and stability of the E3 ubiquitin ligase RING1B are controlled by the ubiquitin system. Self-ubiquitination of RING1B, generating K6, K27 and K48-based mixed polyubiquitin chains, is a prerequisite for its activity as an E3 ligase for histone H2A. Monoubiquitination of histone H2A is one of the hallmarks of Polycomb-mediated gene silencing. The destruction of RING1B however, is mediated through K48 polyubiquitination catalyzed by the ubiquitin ligase E6-AP. Both forms of ubiquitination of RING1B are mutually exclusive and therefore the balance between them may constitute a point of regulation of Polycomb-mediated gene repression. Here we identify ARF as a regulator of RING1B ubiquitination. ARF appears to selectively prevent RING1B self-ubiquitination, probably allowing more efficient E6-AP-mediated ubiquitination and subsequent degradation of RING1B. By binding to the RING domain of RING1B, ARF disrupts RING1B homodimerization, providing a potential mechanism for its effect on RING1B self-ubiquitination.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

PRAJA1 is a ubiquitin ligase for the polycomb repressive complex 2 proteins.

Methylation of lysine 27 on histone H3 by the polycomb repressive complex 2 (PRC2) leads to transcriptional repression of genes which are critical to development. PRC2 core complex is composed of the histone methyltransferase EZH2, EED, and SUZ12. Knockdown of any of the PRC2 core subunits results in a concomitant loss of the other subunits which is mediated by the ubiquitin (Ub)-proteasome system (UPS). Inhibition of cellular methyltransferases by 3-deazaneplanocin A (DZNep) also leads to dissociation of the PRC2 complex and rapid degradation of its subunits. Interestingly, the expression of several Ub ligases was induced following DZNep treatment, suggesting that PRC2 might repress the Ub ligase(s) that target its subunits for degradation. Here we confirm that individual PRC2 subunits are ubiquitinated and rapidly degraded by the proteasome. One of the DZNep-induced Ub ligases, PRAJA1, can target PRC2 subunits for proteasomal degradation. PRAJA1 directly ubiquitinates individual PRC2 subunits in a cell free system, which leads to their proteasomal degradation. Expression of PRAJA1 but not of an inactive RING finger mutant of the protein, enhanced the degradation of individual PRC2 subunits in cells. Taken together, our results suggest a role for PRAJA1 in regulating the level of PRC2 by targeting its free subunits for Ub-mediated proteasomal degradation.

Liver-specific Commd1 knockout mice are susceptible to hepatic copper accumulation.

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(Δhep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(Δhep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(Δhep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(Δhep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(Δhep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(Δhep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(Δhep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.

Regulation of the Polycomb protein RING1B ubiquitination by USP7.

The E3 ubiquitin ligase RING1B plays an important role in Polycomb-mediated gene silencing by monoubiquitinating histone H2A. Both the activity and stability of RING1B are controlled by ubiquitination in two distinct manners. Self ubiquitination of RING1B generates K6, K27 and K48-based mixed polyubiquitin chain, and is required for its activity as a ligase. On the other hand, its proteasomal degradation is mediated by another ligase; E6-AP catalyzes the formation of K48-based chains. Since these two modes of ubiquitination target the same lysine residues and are therefore mutually exclusive, an important mode of regulation of RING1B should be at the level of deubiquitination. Here we identify USP7 as a deubiquitinating enzyme that regulates the ubiquitination state of RING1B. RING1B interacts with USP7, which is mediated in part by its RING domain. In addition, USP7 was found in a complex with other Polycomb proteins, suggesting a broad role in regulating these complexes. Although, USP7 directly and specifically deubiquitinates RING1B in vitro and in vivo, it does not discriminate between the activating and proteolysis-targeting modes of ubiquitination, and therefore has a stabilizing effect on RING1B.

Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome.

The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, "noncanonical" polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for "canonical" ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.

Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.

UNLABELLED: Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD. With the exception of p.S1363F, all mutations tested (p.G85V, p.R778L, p.H1069Q, p.C1104F, p.V1262F, p.G1343V, and p.S1363F) resulted in reduced ATP7B protein expression, whereas messenger RNA abundance was unaffected. Retention of mutant ATP7B in the endoplasmic reticulum, increased protein expression, and normalization of localization after culturing cells at 30 degrees C, and homology modeling suggested that these proteins were misfolded. Four distinct mutations exhibited residual copper export capacity, whereas other mutations resulted in complete disruption of copper export by ATP7B. Treatment with pharmacological chaperones 4-phenylbutyrate (4-PBA) and curcumin, a clinically approved compound, partially restored protein expression of most ATP7B mutants. CONCLUSION: These findings might enable novel treatment strategies in WD by directly enhancing the protein expression of mutant ATP7B with residual copper export activity. 1795.).

Distinct Wilson's disease mutations in ATP7B are associated with enhanced binding to COMMD1 and reduced stability of ATP7B.

BACKGROUND & AIMS: Wilson's disease (WD) is characterized by hepatic copper overload and caused by mutations in the gene encoding the copper-transporting P-type adenosine triphosphatase (ATPase) ATP7B. ATP7B interacts with COMMD1, a protein that is deleted in Bedlington terriers with hereditary copper toxicosis. Here we characterized the implications of the interaction between COMMD1 and ATP7B in relation to the pathogenesis of WD. METHODS: Glutathione-S-transferase pull-down experiments, co-immunoprecipitations, immunofluorescence microscopy, site-directed mutagenesis, and biosynthetic labeling experiments were performed to characterize the interaction between COMMD1 and ATP7B and the effects of WD causing mutations. RESULTS: COMMD1 specifically interacted with the amino-terminal region of ATP7B. This interaction was independent of intracellular copper levels and of the expression of the copper chaperone ATOX1. Four WD patient-derived mutations in this region of ATP7B significantly increased its binding to COMMD1. Two of these mutations also resulted in mislocalization and increased degradation rate of ATP7B. Although COMMD1 did not affect copper-induced trafficking of ATP7B, it markedly decreased the stability of newly synthesized ATP7B. CONCLUSIONS: Our data implicate COMMD1 in the pathogenesis of WD and indicate that COMMD1 exerts its regulatory role in copper homeostasis through the regulation of ATP7B stability.

Characterization of COMMD protein-protein interactions in NF-kappaB signalling.

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-kappaB (nuclear factor kappaB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-kappaB signalling through characterization of protein-protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-kappaB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-kappaB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-kappaB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IkappaBalpha (inhibitory kappaBalpha), indicating that both proteins inhibit NF-kappaB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein-protein interactions and provide new mechanistic insight into the function of this protein family in NF-kappaB signalling.

Bleomycin has antiviral properties against drug-resistant HIV strains and sensitises virus to currently used antiviral agents.

In this study we performed phenotypic assays to assess involvement of the cancer chemotherapeutic agent bleomycin (BLM) in replication inhibition of mutant HIV-1 viral strains. Three clinically relevant mutant HIV variants, including one containing the Q151M mutation conferring multinucleoside resistance, were equally as sensitive to BLM as the wild-type HXB2 strain. Long-term incubation of BLM with a wild-type HIV(Ba-L) strain did not alter the sensitivity of the strain to BLM (IC(50) of BLM 0.64 microM at the beginning of incubation to 0.58 microM). At the same point in time, resistance to lamivudine (3TC) and zidovudine (AZT) was noted. Interestingly, the BLM-treated virus showed hypersensitivity to both AZT and 3TC. Our results suggest a contribution of BLM in viral load reduction in patients receiving both anticancer and antiviral agents and harbouring both wild-type and resistant HIV strains.

Mechanism of inhibition of the human immunodeficiency virus type 1 by the oxygen radical generating agent bleomycin.

Alternative targets of attack of the human immunodeficiency virus (HIV) are necessary in light of infection persistence due to onset of resistance after conventional reverse transcriptase and protease inhibitor therapy. We have recently shown that the cancer chemotherapeutic agent bleomycin (BLM) dose-dependently inhibits HIV-1 replication. The mechanism of this viral inhibition in vitro was investigated. Cell-free wild-type virions were affected directly by BLM in the presence of H2O2, as shown by a 38% decrease of viral infectivity. Viral inhibition by BLM did not proceed via NF-kappaB inhibition. The viral R/U5 DNA product was reduced by 70% without any effect on reverse transcriptase activity. In both a cell-free system as well as two-cell systems the antiviral dependence of BLM on iron and oxidant species was demonstrated. Bleomycin seems to inhibit HIV-1 replication through the same properties that make it a suitable anti-cancer agent. The results presented in this study describe a novel mechanism of HIV-1 inhibition with potential application in viral infections. The anti-HIV effects of BLM in patients receiving this drug in combination with HAART should be carefully monitored in order to evaluate the clinical significance of the findings described in this study.