Two Allelic Variants of Aldo-Keto Reductase 1A1 Exhibit Reduced in Vitro Metabolism of Daunorubicin

ABSTRACT: Aldo-keto reductases (AKRs) are a class of NADPH-dependent oxidoreductases that have been linked to metabolism of the anthracyclines doxorubicin (DOX) and daunorubicin (DAUN). Although widely used, cardiotoxicity continues to be a serious side effect that may be linked to metabolites or reactive intermediates generated in their metabolism. In this study we examine the little known effects of nonsynonymous single nucleotide polymorphisms of human AKR1A1 on the metabolism of these drugs to their alcohol metabolites. Expressed and purified from bacteria using affinity chromatography, the AKR1A1 protein with a single histidine (6x-His) tag exhibited the greatest activity using two test substrates: p-nitrobenzaldehyde (5.09 ؎ 0.16 mol/min/mg of purified protein) and DL-glyceraldehyde (1.24 ؎ 0.17 mol/min/mg). These activities are in agreement with published literature values of nontagged human AKR1A1. The 6x-His-tagged AKR1A1 wild type and allelic variants, E55D and N52S, were subsequently examined for metabolic activity using DAUN and DOX. The tagged variants showed significantly reduced activities (1.10 ؎ 0.42 and 0.72 ؎ 0.47 nmol of daunorubicinol (DAUNol) formed/min/mg of purified protein for E55D and N52S, respectively) compared with the wild type (2.34 ؎ 0.71 nmol/min/mg). The wild type and E55D variant metabolized DOX to doxorubicinol (DOXol); however, the levels fell below the limit of quantitation (25 nM). The N52S variant yielded no detectable DOXol. A kinetic analysis of the DAUN reductase activities revealed that both amino acid substitutions lead to reduced substrate affinity, measured as significant increases in the measured K m for the reduction reaction by AKR1A1. Hence, it is possible that these allelic variants can act as genetic biomarkers for the clinical development of DAUN-induced cardiotoxicity

Biomarkers for Severity of Spinal Cord Injury in the Cerebrospinal Fluid of Rats

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage

Etudes du developpement de la drosophile: variations on a theme

As a holometabolous organism which has been highly exploited genetically, the fruitfly, Drosophila, is an excellent model for the study of development in higher organisms. Conditional mutations that are sensitive to temperature differences were used to investigate problems of gene action in different tissues, regulation of specific tissue determination and differentiation, and the genetic regulation of development and functional integration of the nervous system. The first problem, a classical question in developmental biology, attempts to determine whether control of the activity of a structural gene is directly imparted by the tissue in which the gene product is active or whether, in fact, a freely flowing evocator of gene action exists. A temperature-sensitive ras allele has an effective lethal phase at 29°C about 12 hours after pupation and a temperature-sensitive period (TSP) for lethality beginning midway through the third larval instar and ending around pupation. The mutation also alters the quantity of pteridines present in the eyes, testes and malpighian tubules at 29°C. The TSP for pigment production in the malpighian tubules occurs in the egg and first instar larvae, and in eyes and testes after pupation. The demonstrated autonomy of the mutant in the eyes implies the tissue-specific functioning of the gene. It is suggested that the different TSPs for a single mutation indicate tissue-specific activation of a gene at different times during development, although the possibility of activation of preformed polypeptides has not been eliminated. There exists in Drosophila a class of mutations called "homeotic" which cause changes in determination of the imaginal discs. The second problem investigated concerns the possibility of isolating a temperature-sensive homeotic mutant for the purpose of studying genes which regulate specific pathways of differentiation. The homeotic mutant, ss[sup a40a], was found to have a temperature-sensitive transformation of the arista segment of the antennal complex to a tarsus of the leg. In a selected stock, penetrance was complete so that at 29°C, normal aristae were produced, whereas at 17°C, complete tarsi developed in all flies. "Shift" studies revealed a temperature-sensitive period in the third larval instar. The temperature-initiated action of ss[sup a40a] does not appear to act on a ts receptor site within the disc cells. In combination with another homeotic mutant, Antenna-pedia, the entire antennal complex is transformed to a complete leg at 22°C. Mutants affecting the nervous system were sought for the purpose of investigating the genetic regulation of the development and function of the nervous system. A temperature-sensitive mutation, para[sup ts] of Drosophila melanogaster causes an immediate but reversible paralysis only of adults when shifted from 22°C to 29°C. The mutation is a sex-linked recessive mapping 2.8 units to the left of f. Wild-type flies observed for two hour periods exhibited normal mobility at all temperatures between 22° and 35°C. From 22° to 25°C, para[sup ts] flies were wild-type in walking, climbing and flying ability. At one degree intervals above 25°C, para[sup ts] flies became increasingly debilitated and at 29°C, complete paralysis occurred. After prolonged maintenance at 29°C, recovery of some activity could occur at that temperature. Extensive studies of behavior of mosaics at 29°C revealed a requirement of the + allele in the head for mobility and a thoracic component for proper leg movement. Normal electroretino-grams were obtained at both 22° and 30°C. The results suggest a temperature-sensitive defect in the central nervous system.Science, Faculty ofZoology, Department ofGraduat

Telomeric position effect--a third silencing mechanism in eukaryotes.

Eukaryotic chromosomes terminate in telomeres, complex nucleoprotein structures that are required for chromosome integrity that are implicated in cellular senescence and cancer. The chromatin at the telomere is unique with characteristics of both heterochromatin and euchromatin. The end of the chromosome is capped by a structure that protects the end and is required for maintaining proper chromosome length. Immediately proximal to the cap are the telomere associated satellite-like (TAS) sequences. Genes inserted into the TAS sequences are silenced indicating the chromatin environment is incompatible with transcription. This silencing phenomenon is called telomeric position effect (TPE). Two other silencing mechanisms have been identified in eukaryotes, suppressors position effect variegation [Su(var)s, greater than 30 members] and Polycomb group proteins (PcG, approximately 15 members). We tested a large number of each group for their ability to suppress TPE [Su(TPE)]. Our results showed that only three Su(var)s and only one PcG member are involved in TPE, suggesting silencing in the TAS sequences occurs via a novel silencing mechanism. Since, prior to this study, only five genes have been identified that are Su(TPE)s, we conducted a candidate screen for Su(TPE) in Drosophila by testing point mutations in, and deficiencies for, proteins involved in chromatin metabolism. Screening with point mutations identified seven new Su(TPE)s and the deficiencies identified 19 regions of the Drosophila genome that harbor suppressor mutations. Chromatin immunoprecipitation experiments on a subset of the new Su(TPE)s confirm they act directly on the gene inserted into the telomere. Since the Su(TPE)s do not overlap significantly with either PcGs or Su(var)s, and the candidates were selected because they are involved generally in chromatin metabolism and act at a wide variety of sites within the genome, we propose that the Su(TPE) represent a third, widely used, silencing mechanism in the eukaryotic genome