Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3.

Enhanced version

This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.     

Methylphenidate is not clastogenic in cultured human lymphocytes and in the mouse bone-marrow micronucleus test

Methylphenidate (MPH) is one of the most frequently prescribed drugs for the treatment of attention deficit hyperactivity disorder (ADHD). A report on cytogenetic effects observed in peripheral lymphocytes from children treated for 3 months with MPH raised questions about the genetic toxicity of this compound. A critical review of this data concluded that the cytogenetic effects in treated children remain unexplained. A literature review showed that MPH was found negative in most genetox studies performed, but no in vitro chromosome aberration data in human lymphocytes have been published. Therefore, we conducted a chromosomal aberration study in cultured human peripheral lymphocytes. The results of this investigation showed that d,l-methylphenidate (MPH, Ritalin) in concentrations up to 10 mM did neither induce structural nor numerical chromosome abnormalities. An oral mouse bone-marrow micronucleus test in B6C3F(1) mice, with doses up to 250 mg/kg bw, was negative too. The data of these studies confirm the absence of clastogenic activity of MPH in non-clinical studies.     

Serum Levels of TNF Receptor Ligands Are Dysregulated in Sepsis and Predict Mortality in Critically Ill Patients

IntroductionTNF superfamily members, including TNF-related weak inducer of apoptosis (TWEAK) and Glucocorticoid-Induced TNFR-Related Protein Ligand (GITRL) have been described as serum based biomarkers for inflammatory and immune mediated diseases. However, up to now the role of TWEAK and GITRL has not been analyzed in critical illness and sepsis.MethodsGITRL and TWEAK serum concentrations were measured in 121 critically ill patients (84 fulfilled with septic disease), in comparison to 50 healthy controls. Results were correlated with clinical data.ResultsSerum levels of TWEAK and GITRL were strongly decreased in critically ill patients compared with healthy controls. Concentrations of TWEAK (but not GITRL) were further decreased in patients with sepsis and correlated with routinely used markers of inflammation and bacterial infection such as C-reactive protein, procalcitonin and Interleukin-6. Notably, we failed to detect a correlation to other TNFR ligands such as TNF or APRIL. Finally, TWEAK levels of the upper quartile of the cohort were prognostic for mortality during ICU treatment.ConclusionTWEAK and GITRL levels were lower in intensive care unit medical patients. Levels of TWEAK were further decreased in septic patients, and alterations in TWEAK concentrations were linked to an unfavorable outcome. Together with recently published results on other TNFR ligands, these data indicate specific functions of the different TNFR ligands in septic diseases.     

Decreased Plasma and Cerebrospinal Fluid Ascorbate Levels in Patients With Septic Encephalopathy

Septic encephalopathies rapidly affect brain function without the involvement of a specific area causing a broad range of reversible neurologic symptoms. Capillary leakage including dysfunction of the blood-brain barrier has been proposed as a potential pathogenic mechanism in this entity. We tested the hypothesis that oxidative stress measured in plasma and cerebrospinal fluid (CSF) of patients suffering from septic encephalopathy could be linked to the neurologic symptoms of the disease. The neurologic symptoms of eleven patients with septic encephalopathy were described semiquantitatively through a score system. The ascorbate levels were significantly lower in both plasma and CSF from patients with septic encephalopathy than controls, and in CSF but not plasma this decrease correlated with the severity of neurologic symptoms. No significant changes were found for &#102 -tocopherol. Our findings suggest that the short-term oxidative stress may be an important factor in the development of septic encephalopathy, possibly through dysregulation of the blood-brain barrier.     

The influence of smoking on plasma folate and lipoproteins in Alzheimer disease, mild cognitive impairment and depression

The risk for Alzheimer's disease (AD) is associated with lifestyle factors, especially cigarette smoking. In this study we investigated the influence of smoking on the serum levels of folic acid, LDL and HDL in AD patients, patients with minimal cognitive impairment (MCI) and patients with major depression. We investigated a total of n = 374 patients in the diagnostic categories:, AD: n = 272, MCI: n = 60, Major depression: n = 42. We found significantly lower HDL levels in smokers and previous smokers in comparison to non-smokers, p<0,05. The LDL: HDL ratio in smokers was significant higher (+20%) compared to previous smokers and non-smokers, p < 0.05. The mean levels of folic acid were statistically significant (p<0.05) lower (-24%) in smokers compared to non-smokers. Patients with MCI and Alzheimer;s disease (and also major depression) who are "smokers" show serum levels of HDL and folic acid that are known to be strong risk factors for vascular damage and increased risk for vascular brain damage and impaired cognitive function. Therefore cessation of smoking, substitution with folate or statin therapy of smoking patients with MCI or AD might be beneficial to slow down further cognitive decline.     

The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.     

Vitamin B 12 plasma concentrations in Alzheimer disease

In this contribution we investigated the correlation between plasma vitamin B12 levels and cognitive impairment in Alzheimer's disease. We could demonstrate a significant inverse correlation when the MMSE scores of those patients with the lowest 10% Vitamin B12 plasma levels (<184 ng/ml) were compared with the upper 10% Vitamin B12 plasma levels (>598 ng/ml): p=0.008, Spearman-Rho= -0.36. MMSE in the upper percentile of plasma B12 levels was 20.0 +/- 4.6 and in the lower percentile 15.7 +/- 6.1, resulting in a difference of 4.3 MMSE points. We conclude, that vitamin B12 deficiency could aggravate or accelerate the course of Alzheimer disease as vitamin B12 possesses neuroprotective and anti-inflammatory properties.     

Induction of A:T to G:C transition mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue, in the bone marrow of Muta Mouse

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

The roles of carnosine in aging of skeletal muscle and in neuromuscular diseases

Skeletal muscles undergo specific alterations that are related to the aging process. The incidence of several neuromuscular diseases (e.g., amyotrophic lateral sclerosis (ALS), myasthenia gravis, polymyositis, drug-induced myopathies, late-onset mitochondrial myopathy) is age-related. The increased sensitivity to disease of aging muscle represents an additional age-related negative influence in the presence of existing risk factors (such as a genetic predisposition). The potential significance of carnosine lies on one hand in its possible influence on specific physiological changes in muscle associated with the aging process, and on the other in its effect on oxidative stress and the antioxidative system in specific neuromuscular diseases such as ALS or polymyositis.