The Natural History of Severe Acute Liver Injury.

OBJECTIVES: Acute liver failure (ALF) is classically defined by coagulopathy and hepatic encephalopathy (HE); however, acute liver injury (ALI), i.e., severe acute hepatocyte necrosis without HE, has not been carefully defined nor studied. Our aim is to describe the clinical course of specifically defined ALI, including the risk and clinical predictors of poor outcomes, namely progression to ALF, the need for liver transplantation (LT) and death. METHODS: 386 subjects prospectively enrolled in the Acute Liver Failure Study Group registry between 1 September 2008 through 25 October 2013, met criteria for ALI: International Normalized Ratio (INR)≥2.0 and alanine aminotransferase (ALT)≥10 × elevated (irrespective of bilirubin level) for acetaminophen (N-acetyl-p-aminophenol, APAP) ALI, or INR≥2.0, ALT≥10x elevated, and bilirubin≥3.0 mg/dl for non-APAP ALI, both groups without any discernible HE. Subjects who progressed to poor outcomes (ALF, death, LT) were compared, by univariate analysis, with those who recovered. A model to predict poor outcome was developed using the random forest (RF) procedure. RESULTS: Progression to a poor outcome occurred in 90/386 (23%), primarily in non-APAP (71/179, 40%) vs. only 14/194 (7.2%) in APAP patients comprising 52% of all cases (13 cases did not have an etiology assigned; 5 of whom had a poor outcome). Of 82 variables entered into the RF procedure: etiology, bilirubin, INR, APAP level and duration of jaundice were the most predictive of progression to ALF, LT, or death. CONCLUSIONS: A majority of ALI cases are due to APAP, 93% of whom will improve rapidly and fully recover, while non-APAP patients have a far greater risk of poor outcome and should be targeted for early referral to a liver transplant center.

D-MELD, a simple predictor of post liver transplant mortality for optimization of donor/recipient matching.

Numerous donor and recipient risk factors interact to influence the probability of survival after liver transplantation. We developed a statistic, D-MELD, the product of donor age and preoperative MELD, calculated from laboratory values. Using the UNOS STAR national transplant data base, we analyzed survival for first liver transplant recipients with chronic liver failure from deceased after brain death donors. Preoperative D-MELD score effectively stratified posttransplant survival. Using a cutoff D-MELD score of 1600, we defined a subgroup of donor-recipient matches with significantly poorer short- and long-term outcomes as measured by survival and length of stay (LOS). Avoidance of D-MELD scores above 1600 improved results for subgroups of high-risk patients with donor age >/=60 and those with preoperative MELD >/=30. D-MELD >/=1600 accurately predicted worse outcome in recipients with and without hepatitis C. There is significant regional variation in average D-MELD scores at transplant, however, regions with larger numbers of high D-MELD matches do not have higher survival rates. D-MELD is a simple, highly predictive tool for estimating outcomes after liver transplantation. This statistic could assist surgeons and their patients in making organ acceptance decisions. Applying D-MELD to liver allocation could eliminate many donor/recipient matches likely to have inferior outcome.

Hereditary hemochromatosis.

Hereditary hemochromatosis (HH) refers to several inherited disorders of iron metabolism leading to tissue iron overload. Classical HH is associated with mutations in HFE (C282Y homozygotes or C282Y/H63D compound heterozygotes) and is almost exclusively found in populations of northern European descent. Non-HFE associated HH is caused by mutations in other recently identified genes involved in iron metabolism. Hepcidin is an iron regulatory hormone that inhibits ferroportin-mediated iron export from enterocytes and macrophages. Defective hepcidin gene expression or function may underlie most forms of HH. Target organs and tissues affected by HH include the liver, heart, pancreas, joints, and skin, with cirrhosis and diabetes mellitus representing late signs of disease in patients with markedly elevated liver iron concentration. Compound heterozygotes have milder disease than C282Y homozygotes and clinical signs of HH in these patients are usually associated with other factors such as alcoholism and the dysmetabolic syndrome. The most frequent causes of death in HH are liver cancer, cirrhosis, cardiomyopathy, and diabetes, but patients who undergo successful iron depletion before the development of cirrhosis or diabetes can have normal survival. Classical HH is characterized by incomplete penetrance and variable expressivity, and women are less affected than men by iron overload and iron overload-related disease. The diagnosis of HH is established by genetic testing in patients with elevated transferrin saturation values. Patients with an established diagnosis of HH and iron overload should be treated with phlebotomy to achieve body iron depletion followed by maintenance phlebotomy. Population screening for HH is controversial principally because of incomplete penetrance, but screening of selected, high risk populations and first-degree relatives of affected probands may be cost effective.

The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner.

An essential aspect of progression through mitosis is the sequential degradation of key mitotic regulators in a process that is mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase [1]. In mitotic cells, two forms of the APC/C exist, APC/C(Cdc20) and APC/C(Cdh1), which differ in their associated WD-repeat proteins (Cdc20 and Cdh1, respectively), time of activation, and substrate specificity [2, 3]. How the WD-repeat proteins contribute to APC/C's activation and substrate specificity is not clear. Many APC/C substrates contain a destruction box element that is necessary for their ubiquitination [4-6]. One such APC/C substrate, the budding yeast anaphase inhibitor Pds1 (securin), is degraded prior to anaphase initiation in a destruction box and APC/C(Cdc20)-dependent manner [3, 7]. Here we find that Pds1 interacts directly with Cdc20 and that this interaction requires Pds1's destruction box. Our results suggest that Cdc20 provides a link between the substrate and the core APC/C and that the destruction box is essential for efficient Cdc20-substrate interaction. We also find that Pds1 does not interact with Cdh1. Finally, the effect of spindle assembly checkpoint activation, known to inhibit APC/C function [8], on the Pds1-Cdc20 interaction is examined.

Sister chromatid separation: falling apart at the seams.

Cohesion between sister chromatids must be dissolved at the time of chromosome segregation. Recent studies reveal that the principles of cohesion dissolution in mitosis and meiosis are the same, but that there are important differences that stem from the distinct natures of these two processes.

Pds1p of budding yeast has dual roles: inhibition of anaphase initiation and regulation of mitotic exit.

Progression through mitosis is controlled by protein degradation that is mediated by the anaphase-promoting complex/cyclosome (APC/C) and its associated specificity factors. In budding yeast, APC/C(Cdc20) promotes the degradation of the Pds1p anaphase inhibitor at the metaphase-to-anaphase transition, whereas APC/C(Cdh1) promotes the degradation of the mitotic cyclins at the exit from mitosis. Here we show that Pds1p has a novel activity as an inhibitor of mitotic cyclin destruction, apparently by preventing the activation of APC/C(Cdh1). This activity of Pds1p is independent of its activity as an anaphase inhibitor. We propose that the dual role of Pds1p as an inhibitor of anaphase and of cyclin degradation allows the cell to couple the exit from mitosis to the prior completion of anaphase. Finally, these observations provide a novel regulatory paradigm in which the sequential degradation of two substrates is determined by the substrates themselves, such that an early substrate inhibits the degradation of a later one.

The metaphase to anaphase transition: a case of productive destruction.

The metaphase to anaphase transition is a point of no return; the duplicated sister chromatids segregate to the future daughter cells, and any mistake in this process may be deleterious to both progeny. At the heart of this process lies the anaphase inhibitor, which must be degraded in order for this transition to take place. The degradation of the anaphase inhibitor occurs via the ubiquitin-degradation pathway, and it involves the activity of the cyclosome/anaphase promoting complex (APC). The fidelity of the metaphase to anaphase transition is ensured by several different regulatory mechanisms that modulate the activity of the cyclosome/APC. Great advancements have been made in this field in the past few years, but many questions still remain to be answered.

The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway.

Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct. We have previously shown that the S. cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis.

The metaphase-to-anaphase transition: avoiding a mid-life crisis.

The metaphase-to-anaphase transition is a highly regulated process, which is governed by the activity of the anaphase-promoting complex (APC). The APC promotes the degradation of several proteins, including mitotic cyclins and newly identified anaphase inhibitors. Several discoveries made this year shed invaluable light on the regulation of APC activation and its substrate specificity.

Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p.

Anaphase initiation has been postulated to be controlled through the ubiquitin-dependent proteolysis of an unknown inhibitor. This process involves the anaphase promoting complex (APC), a specific ubiquitin ligase that has been shown to be involved in mitotic cyclin degradation. Previous studies demonstrated that in Saccharomyces cerevisiae, Pds1 protein is an anaphase inhibitor and suggested that it may be an APC target. Here we show that in yeast cells and in mitotic Xenopus extracts Pds1p is degraded in an APC-dependent manner. In addition, Pds1p is directly ubiquitinated by the Xenopus APC. In budding yeast Pds1p is degraded at the time of anaphase initiation and nondegradable derivatives of Pds1p inhibit the onset of anaphase. We conclude that Pds1p is an anaphase inhibitor whose APC-dependent degradation is required for the initiation of anaphase.

Reconstitution of repair-gap UV mutagenesis with purified proteins from Escherichia coli: a role for DNA polymerases III and II.

Using a cell-free system for UV mutagenesis, we have previously demonstrated the existence of a mutagenic pathway associated with nucleotide-excision repair gaps. Here, we report that this pathway can be reconstituted by using six purified proteins: UvrA, UvrB, UvrC, DNA helicase II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, although less effectively, whereas DNA polymerase I, the major repair polymerase, could not. DNA sequence analysis of mutations generated in the in vitro reaction revealed a spectrum typical of mutations targeted to UV lesions. These observations suggest that repair-gap UV mutagenesis is performed by DNA polymerase III, and to a lesser extent by DNA polymerase II, by filling-in of a rare class of excision gaps that contain UV lesions.

Deamination of cytosine-containing pyrimidine photodimers in UV-irradiated DNA. Significance for UV light mutagenesis.

The realization that cytosine in cyclobutyl pyrimidine dimers rapidly deaminates to uracil raised the possibility that this chemical transformation, rather than an enzymatic polymerase error, is the major mutagenic step in UV mutagenesis. We have established a sensitive bioassay system that enabled us to determine the rate of deamination of cytosine in cyclobutyl pyrimidine dimers in plasmid DNA. This was done by in vitro UV irradiation and deamination of a plasmid carrying the cro gene, followed by photoreactivation, and assaying uracils in DNA by their ability to cause Cro- mutations in an indicator strain that was deficient in uracil DNA N-glycosylase. DNA sequence analysis revealed that 27 out of 29 Cro- mutants carried GC --> AT transitions, as expected from deamination of cytosine. Deamination of cytosines in the cro gene in UV-irradiated plasmid pOC2 proceeded at 37 degrees C with first-order kinetics, at a rate of (3.9 +/- 0.6) x 10(-5) s-1, corresponding to a half-life of 5 h. Physiological salt conditions increased the half-life to 12 h, whereas decreasing the pH increased deamination. The temperature dependence of the rate constant yielded an activation energy of 13.6 +/- 3.3 kcal/mol. These kinetics data suggest that deamination of cytosine-containing dimers is too slow to play an important role in UV mutagenesis in Escherichia coli. However, it is likely to play an important role in mammalian cells, where the mutagenic process is slower.

In vitro UV mutagenesis associated with nucleotide excision-repair gaps in Escherichia coli.

Using a cell-free system for UV mutagenesis we have recently shown that extracts prepared from Escherichia coli cells promote a UV mutagenesis pathway that depends on the uvrABC repair genes independent of DNA replication (type II UV mutagenesis; Cohen-Fix, O., and Livneh, Z. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3300-3304). Type II UV mutagenesis was defective also in extracts prepared from a uvrD strain. These deficiencies were complemented by adding purified UvrA, UvrB, UvrC, or UvrD proteins to the respective cell extracts. The Uvr proteins act at an early stage in the process, probably preparing a premutagenic single-stranded DNA gap, which subsequently serves as a substrate for the mutagenic reaction. Type II UV mutagenesis was not dependent on DNA polymerases I or on DNA polymerase II, but it was dependent on DNA polymerase III. Thus, similar to the in vivo situation, only DNA polymerase III is essential for UV mutagenesis. Antibodies against the beta subunit of DNA polymerase III holoenzyme inhibited DNA replication but not UV mutagenesis. Thus, the processivity subunit of the holoenzyme is not required for type II UV mutagenesis, in agreement with a mechanism involving filling-in of short single-stranded DNA gaps.

Replication of damaged DNA and the molecular mechanism of ultraviolet light mutagenesis.

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway.

Incubation of UV-irradiated plasmid DNA with a protein extract prepared from Escherichia coli cells led to the production of mutations in the cro gene residing on the plasmid. The mutations were detected in a subsequent bioassay step, which involved transformation of an indicator strain with the plasmid DNA that was retrieved from the reaction mixture, followed by plating on lactose/MacConkey plates. UV mutations produced in this cell-free reaction required the recA and umuC gene products and were prevented by rifampicin, an inhibitor of RNA polymerase, which inhibited plasmid replication. Removal of pyrimidine photodimers from the plasmid by enzymatic photoreactivation after the in vitro stage, but prior to transformation, increased plasmid survival as expected. Surprisingly, it also caused a large increase in the frequency of UV mutations detected in the bioassay. This photoreactivation-stimulated in vitro UV mutagenesis was dependent on the excision repair genes uvrA, uvrB, and uvrC and occurred in the absence of DNA replication. This suggests that two distinct UV mutagenesis pathways occurred in vitro: a replication-dependent pathway (type I) and a repair-dependent pathway (type II). DNA sequence analysis of type II UV mutations revealed a spectrum similar to that of in vivo UV mutagenesis. When the photoreactivation step was included in the protocol, type II UV mutagenesis did not require the RecA and UmuC proteins. These results are in agreement with the in vivo delayed photoreactivation phenomenon, where the removal of photodimers after an incubation period eliminated the requirement for RecA and UmuC in UV mutagenesis. The above system will enable the biochemical analysis of UV mutagenesis and the isolation of proteins involved in the process.