Various preservative-free eye drop formulations for glaucoma treatment have been marketed intending to decrease ocular surface side effects and improve tolerability. However, preservative-free eye drops including different solubilizers to dissolve the antiglaucoma drugs may induce detrimental effects in the eye. In this study, we exposed human corneal epithelial cells (HCE-2) for 1, 6, 12, 24 and 48 hours to the first preservative-free (PF) tafluprost (Taflotan®), the recently-launched preservative-free (PF) latanoprost (Monoprost®), preservative benzalkonium chloride (BAK) and the excipient macrogolglycerol hydroxystearate 40 (MGHS40) using dilutions 0.1%, 0.3%, 1.0%, 3.0% and 10.0% of the original products. The cells also were exposed to undiluted PF tafluprost and PF latanoprost once a day for 9 days. Cellular morphology was examined by light microscopy and cell proliferation by Ki-67 fluorescent staining with cell viability being determined by erythrosine staining and the release of lactate dehydrogenase (LDH). Mitochondrial metabolic activity was evaluated with the colorimetric MTT assay. The secretion of interleukin 6 (IL-6) was measured with ELISA. HCE-2 cells displayed no significant morphological changes after PF tafluprost treatment, but PF latanoprost caused clear cell loss. Moreover, PF latanoprost, BAK and MGHS40 evoked cellular damage and inflammation with increasing concentrations and time. Furthermore, undiluted daily PF latanoprost application significantly increased LDH release and IL-6 secretion as compared to PF tafluprost. MGHS40 was observed to be associated with the toxicity of PF latanoprost. Excipients in ocular drops should receive more attention in the future, since they seem to trigger similar detrimental effects in cells as preservatives.
Synaptophysin is a specific presynaptic marker for neurons. Loss of synaptophysin occurs in Parkinson's disease, dementia with Lewy bodies and other neurodegenerative diseases. In vitro studies on synaptophysin are important to understand both the function of the protein itself and its implication in the pathogenesis of neurological diseases. In this study, we determined synaptophysin protein expression by Western analysis in 6 different dopaminergic cell lines including one human (SH-SY5Y), two rat (PC12 and N27) and 3 mouse (MN9D, Cath.a and CAD) cell lines. We found that synaptophysin protein is richly expressed in PC12 cells, but much less in other cells we studied. The order of synaptophysin expression from high to low for the other 5 cell lines was CAD> SH-SY5Y> MN9D> Cath.a = N27 cells, with Cath.a and N27 cells expressing almost undetectable content of synaptophysin protein. These data may be useful to other researchers in choosing a dopaminergic cell line as a model system to study the pathophysiology of neuron terminal loss.
Many studies have shown that compensatory mechanisms are an important aspect of disease progression in both experimental Parkinson's disease (PD) models as well as in PD patients, including compensation by dopaminergic [1-3] and nondopaminergic pathways such as acetylcholinergic [4], GABAergic [4], and glutamatergic [5] systems. For example, when dopamine levels decrease, synthesis of dopamine is increased promptly by rapid upregulation of tyrosine hydroxylase (TH) activity [2]. After this initial response, slower compensatory mechanisms take effect. These mechanisms include increased TH expression as well as increased sensitivity of striatal neurons to dopamine, the latter is caused by augmented sensitivity or by increased expression of postsynaptic dopamine receptors [1, 3]. In addition, Bezard and Gross [6] postulated that a correlation exists between defined stages of PD and specific compensatory mechanisms. Development of strategies that build upon these compensatory effects, particularly at early stages of the disease, can help maintain or possibly regain normal motor function in PD patients by enabling the survival of remaining dopaminergic neurons. These strategies may be crucial for disease treatment. A recently published study in the Journal of Neuroscience by Golden et al. [7] reported that congenital loss of dopaminergic neurons induces a remarkable adaption of the nigrostriatal system, which allows a limited amount of striatal dopamine to maintain normal motor function. This study highlights the potential for the activation of compensatory systems as promising therapies that would help PD patients adapt to the loss of dopamine, alleviating their motor symptoms. Golden et al. [7] established a mouse model which expresses diphtheria toxin (DTA) in dopamine transporter (DAT)-positive neurons to create a model of developmental damage specific to dopaminergic neurons (DAT-DTA mice). DAT-DTA mice exhibited up to a 90% loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA, Fig. 1D), a significant reduction of dopaminergic fibers in the striatum (Fig. 6), and a concomitant depletion of striatal dopamine (Fig. 7). These pathological features mimic those observed in PD patients. Motor and sensorimotor function in these animals was evaluated by conducting a broad spectrum of behavioral tests including locomotor activity (Fig. 4A-C), rotarod (Fig. 4D-H), pole climbing (Fig. 5A,B), movement-initiation (Fig. 5C), inverted screen test (Fig. 5D), and gait analysis (Fig.5E,F). Surprisingly, almost no motor behavior deficits were detected in DAT-DTA mice. When treated with haloperidol, a dopamine receptor antagonist, performance of DAT-DTA mice in the open-field test and the movement initiation test was impaired to the same degree as control mice (Fig. 8A), indicating that the remaining striatal dopamine (3% of control; Fig. 7A) is sufficient to sustain normal motor function in DAT-DTA mice. To further investigate how small amounts of striatal dopamine sustain normal motor function in DAT-DTA mice, their sensitivity to L-DOPA was measured. A dose of L-DOPA that did not affect control mice in the open-field test significantly increased locomotor activity of DAT-DTA mice (Fig. 8B). These results suggest that striatal dopamine receptors are highly sensitized in DAT-DTA mice, providing a plausible explanation for the observed preservation of motor behavior by very small amounts of dopamine. The studies by Golden et al. [7] have significantly advanced our knowledge and have uncovered new, important avenues of research. Non-motor symptoms including olfaction impairment, autonomic nervous system failure, cognitive dysfunction, and mood disorders precede motor symptoms in PD [8]. Thus, it would be important to determine whether the DAT-DTA mice exhibit non-motor symptoms. For example, dopamine levels are positively associated with learning and memory [9, 10], the severe depletion of dopamine in the DAT-DTA mice could conceivably affect cognitive function. Furthermore, VTA dopaminergic neurons project to prefrontal cortex. Ascertaining whether loss of VTA dopaminergic neurons (Fig. 1D) leads to a decrease in dopamine levels in prefrontal cortex would provide key information related to non-motor function in DAT-DTA mice, as prefrontal dopamine signaling regulates cognitive and executive function. Moreover, the DAT-DTA model may be useful to study vulnerability to PD risk factors such as aging, and exposure to neurotoxins (e.g. rotenone, paraquat, 6-hydroxydopamine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The study of Golden et al. (2013) may also provide key insights into the mechanisms that permit the more efficient utilization of remaining dopaminergic neurons after their depletion in PD. While the studies by Golden et al. [7] have many merits listed above, there are some caveats that might affect their conclusions. For example, one caveat is that most experimental sample sizes are below 10 (n=2-13, Figs 4, 5, 8). In most behavioral studies using mouse models sample sizes of 10-20 are commonly needed to provide sufficient statistical power to detect moderate differences between groups [11]. Another weakness of the study is that male and female animals were pooled for all behavioral tests, but no evidence was provided whether there were any differences in outcomes between genders [11]. Although the authors reported marginal interindividual variance among animals in the various experimental groups, differences could have become apparent with larger sample sizes and/or by separating sexes. Furthermore, body weight is a major confounding factor for many motor behavioral assays used in this study (e.g. rotarod, locomotor activity, pole test, inverted screen, and gait analysis) [12]. While DAT-DTA mice weighed significantly less than age-matched controls (females >3 months; males >10 months, Fig.1B, C), the influence of body weight was not parsed out in the data analysis. Thus, longer latency to fall on the inverted screen for DAT-DTA mice as compared to control mice (Fig. 5D) might be explained solely by their reduced body weight (Fig. 1B, C) [7]. Two more caveats should be considered regarding the progressive loss of dopaminergic neurons in SNc and VTA (Fig. 1D-F). First, the effect of age and genotype and the significance of differences in performance of control and DAT-DTA mice at each of the 4 time points examined would have been best determined using 2-way ANOVA or a mixed model instead of Student's t-test. Second, because defects in rotarod performance become apparent at the age of 18- or 24 months old, studies using animals at those ages (older than 12 months) would be necessary to determine whether there is further loss of dopaminergic neurons. These studies would be very important because they would determine whether there is a threshold for the number of dopaminergic neurons needed before motor deficits are present in this model. Also, a progressive loss of dopaminergic neurons with advanced age could call into question some of the data that combine 18-24 (Fig. 4, 5A-D) and 12-24 months old (Fig. 7) animals [7]. Because of these potential confounds, it is possible that 24 month-old DAT-DTA mice might still be presymptomatic rather than asymptomatic. Because there was no reported difference in motor function between DAT-DTA and control mice at the age tested (Figs. 4, 5), and behavioral data is highly variable, a vehicle-treated DAT-DTA group should have been included in the haloperidol and L-DOPA experiments to make the results more meaningful. This oversight in the experimental design compromised the conclusion that motor behavior in DAT-DTA mice is maintained by dopamine-dependent compensation. Additionally, Golden et al. [7] did not discuss an important study previously published in Trends in Neuroscience, which reported that presymptomatic compensation in PD is not dopamine-mediated [13]. Bezard et al. [13] proposed three functional compensatory changes within and outside of the basal ganglia in the presymptomatic period of PD. In the first period, dopamine homeostatic compensatory mechanisms mask the disease. In the second period, after the breakdown of striatal dopamine homeostasis, a more powerful compensation takes place within the basal ganglia to increase the activity of the basal ganglia output structures (e.g. globus pallidus). In the third period, robust compensation within and outside (e.g. supplementary motor area) the basal ganglia takes place and plays a crucial role in leading to emergence of motor abnormalities. Thus, it is conceivable that DAT-DTA mice may represent a model of the second or third stages of the presymptomatic period as proposed by Bezard et al. Further investigation of the activity of output structures within and outside basal ganglia should address these possibilities.
As an essential organ for gas exchange, the lungs are constantly exposed to the external environment and are simulated by toxicants and pathogens. The integrity of lung epithelium and epithelial cells is crucial for fulfilling the physiological functions of the lung. The homeostasis of lung epithelial cells is maintained by a complex network by which survival and death are tightly regulated. Upon noxious stimulation, lung epithelium attempts to maintain its normal structure and function. Savage of injured cells and clearance of unsalvageable dying cells or unwanted proliferated cells constantly occur in the lung epithelium. Apoptosis, or programmed cell death, functions as a primary mechanism to discard unsalvageable cells or unwanted overgrowth. Autophagy, on the other hand, initially attempts to save and repair the injured cells. However, when the noxious stimulation is too strong and cell survival becomes unfeasible, autophagy behaves oppositely and cooperates with apoptosis, subsequently accelerates cell death. The imbalance between autophagy and apoptosis potentially leads to tumorigenesis or devastating cell death/lung injury. Therefore, the cross-talk between apoptosis and autophagy in lung epithelial cells is critical in determining the fate of epithelial cells and its balance of death/survival in response to environmental stimuli. In this review, we will focus on the current understandings of the communications between apoptosis and autophagy in lung epithelial cells. We will review multiple key regulators and their underlying mechanisms involved in the cross-talk between apoptosis and autophagy. The autophagic factors, such as the Beclin-1, ATG5, Fap-1, p62 and concentration-dependent LC3B, all closely interact with multiple apoptosis pathways. Understanding these regulations of apoptosis/autophagy cross-talk potentially provides novel targets for developing diagnostic and therapeutic strategies for many lung diseases, including lung injuries and malignancies.
Much evidence suggests that oxidative stress plays a role in schizophrenia pathogenesis. Major oxidative stress sources include hydrogen peroxide and biogenic aldehydes that are mainly cleared in vivo by glutathione peroxidase (GPX) and aldehyde dehydrogenase (ALDH), respectively. Both enzymes are richly expressed in brain. Schizophrenia patients have significantly increased plasma levels of malondialdehyde and glutathione, combined with decreased GPX activity and ALDH1 mRNA levels in the ventral tegmental area. Absence of Aldh1a1 (murine homolog of ALDH1) gene causes increased basal extracellular dopamine concentrations, a common characteristic of schizophrenia. Studies investigating association between gene polymorphisms of GPX1 (the most abundant form of GPX) or ALDH1A1 with schizophrenia also have not clearly demonstrated whether ALDH1A1 or GPX1 is involved in pathogenesis of schizophrenia. To investigate possible contributions of ALDH and GPX to pathological behaviors associated with schizophrenia, we generated mice with both Aldh1a1 and Gpx1 gene deletions (KO). Aldh1a1/Gpx1 KO and wild type (WT) mice had similar number of novel entry and alteration in Y-maze test, suggesting no cognition deficit in KO. Furthermore, KO and WT displayed similar social interaction and novelty preferences in three chambered tests. Overall, KO and WT had similar activity levels, as indicated by their entries in the Y-maze and sociability tests. Furthermore both genotypes buried a similar percentage of marbles in a 30 min marble-burying task. In summary, homozygous deletion of Gpx1 and Aldh1a1 genes was not associated with schizophrenia-like behavioral phenotypes including anxiety, hyperactivity, cognitive deficit or social disability. Our findings suggest that constitutive absence of these genes alone is unlikely to give rise to common behavioral schizophrenia symptoms. However, these mice may be highly sensitive to oxidative challenges during critical stages of prenatal or juvenile brain development.
Excess nutrient uptake leads to obesity, insulin resistance, and type 2 diabetes. Mammalian target of the rapamycin (mTOR), a major component of the nutrient-sensing pathway also regulates mitochondrial oxidative function. Rapamycin, a pharmacological inhibitor of mTOR, causes glucose intolerance and inhibits mitochondrial oxidative function. While a number of studies have focused on the effect of rapamycin on control wild-type mice, ours is the first to study the effect of rapamycin on mitochondrial gene expression and insulin sensitivity in the db/db mouse, a model of diabetic dyslipidemia. Female db/+ and db/db mice were fed ad libitum a rapamycin-containing diet or a control diet for 6 months, starting at two months of age. Body weight, fat mass, lean mass and food intake were measured monthly. Effect of rapamycin or control diet on markers of adipogenesis, fatty acid oxidation and mitochondrial biogenesis in the gonadal white adipose tissue (WAT) as well as different serum parameters were assessed. Whole body insulin sensitivity was measured by insulin tolerance test. Rapamycin feeding to db/db mice decreased body weight (58%) and fat mass (33%), elevated markers of fatty acid oxidation and mitochondrial biogenesis in WAT, reduced circulating non-esterified free fatty acids (NEFA), elevated circulating adiponectin and improved insulin sensitivity, compared to control diet fed db/db mice. These data demonstrate that rapamycin exhibits an anti-obesity effect and improves whole body insulin sensitivity in db/db mice and suggest an unexpected effect of simultaneous inhibition mTOR and leptin signaling in mice.
Sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, regulates various physiological functions. We observed that the S1P receptor subtype 1 (S1P1), a high affinity G-protein coupled receptor of S1P, is the major S1P receptor expressed in the Kit+/Sca-1+/Lin- (KSL) hematopoietic stem progenitor cells (HSPCs, KSL-HSPCs). In this study, we investigate function of S1P1 receptors in the regulation of HSPC mobilization in animals. Treatment with SEW2871, a specific agonist of S1P1, had no effect on KSL-HSPC mobilization. In addition, mice pretreated with SEW2871 followed by AMD3100, a well-known activator of KSL-HSPC mobilization by antagonizing the stromal-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling axis, did not enhance the AMD3100-induced KSL-HSPC mobilization. In contrast, pretreatment of (R)-3-amino-4-(3-hexylphenylamino)-4-oxobutyl phosphonic acid (W146), a selective antagonist of S1P1, significantly augments AMD3100-induced KSL-HSPC mobilization into peripheral blood. The inactive enantiomer W140 was incapable of enhancing the AMD3100-induced KSL-HSPC mobilization. Moreover, treatment with selective antagonists for S1P2 and S1P3 had no effects on AMD3100-mediated KSL-HSPC mobilization. Collectively, our data suggest that S1P/S1P1 signaling regulates the SDF-1/CXCR4-mediated retention of KSL-HSPCs in bone marrow microenvironment.
The precise control of gene expression is essential for all biological processes. In addition to DNA-binding transcription factors, numerous transcription cofactors contribute another layer of regulation of gene transcription in eukaryotic cells. One of such transcription cofactors is the highly conserved Mediator complex, which has multiple subunits and is involved in various biological processes through directly interacting with relevant transcription factors. Although the current understanding on the biological functions of Mediator remains incomplete, research in the past decade has revealed an important role of Mediator in regulating lipid metabolism. Such function of Mediator is dependent on specific transcription factors, including peroxisome proliferator-activated receptor-gamma (PPARγ) and sterol regulatory element-binding proteins (SREBPs), which represent the master regulators of lipid metabolism. The medical significance of these findings is apparent, as aberrant lipid metabolism is intimately linked to major human diseases, such as type 2 diabetes and cardiovascular disease. Here, we briefly review the functions and molecular mechanisms of Mediator in regulation of lipid metabolism.
Natural polyphenols, such as resveratrol, have beneficial functions on major human diseases such as cancer, diabetes, and cardiovascular disease. Besides acting as antioxidants, some of these polyphenols can also target proteins to modulate specific biological pathways. The lysine-specific histone demethylase LSD1 plays important roles in cell growth, differentiation and nutrient metabolism. Here, we studied the effect of natural polyphenols resveratrol, curcumin, quercetin and analogs on LSD1. Using in vitro LSD1 enzymatic assays, we show that resveratrol, curcumin and quercetin displayed a potent inhibitory effect on the LSD1 activity and were more potent than the known LSD1 inhibitor trans-2-phenylcyclopropylamine (TCP). The new function of resveratrol, curcumin and quercetin is independent of their antioxidant properties, as other antioxidants had no effect on LSD1 under the similar conditions. In C2C12 fibroblasts, resveratrol and curcumin can efficiently inhibit myogenic expression and differentiation, for which LSD1 is required. Thus, our study has identified LSD1 as a novel target of bioactive natural compounds, such as resveratrol, curcumin and quercetin, and such finding suggests that LSD1 inhibition can at least partially contribute to some of the previously observed beneficial effects of these compounds.
The etiology of lower urinary tract symptoms is poorly understood. The pathophysiology of detrusor instability, voiding dysfunction and pelvic pain in patients with non-obstructed bladder remains highly controversial. In the male, most cases of lower urinary tract symptoms are attributed to bladder outlet obstruction due to benign prostatic hyperplasia. However, urodynamic data have revealed that in approximately one third to more than one half of cases, lower urinary tract symptoms are not associated with enlarged prostate or bladder outlet obstruction. Interestingly, lower urinary tract symptoms questionnaires in women yield scores that are similar to their age-matched male counterparts. These observations imply that aging-associated sex-independent changes in bladder vasculature, nerves, smooth muscle and epithelium may play a role in the development of lower urinary tract symptoms. Epidemiologic studies have shown a close correlation between vascular occlusive disorders and the prevalence of lower urinary tract symptoms. International prostate symptom scores were found to be significantly worse in men with cardiovascular disorders than symptomatic patients without cardiovascular problems. Clinical trials have revealed a close correlation between decreased pelvic blood flow and severity of lower urinary tract symptoms in the elderly patients. Studies with experimental models of pelvic ischemia have shown that accumulation of reactive oxygen species in the ischemic bladder initiates a cascade of cellular, subcellular and molecular reactions. These reactions to ischemia appear to compromise bladder structure and function leading to neurodegeneration, smooth muscle instability, increased contractile activity, fibrosis and non-compliance. These observations collectively introduce a new concept in the pathophysiology of voiding dysfunction suggesting that pelvic ischemia may be an independent factor in the development of non-obstructed non-neurogenic overactive bladder and lower urinary tract symptoms.
Mammalian cells and tissues respond to chemical and physical stress by inducing adaptive or protective mechanisms that prolong survival. Among these, the major stress inducible proteins (heat shock proteins, glucose regulated proteins, heme oxygenase-1) provide cellular protection through protein chaperone and/or anti-oxidative and anti-inflammatory functions. In recent years it has become clear that autophagy, a genetically-programmed and evolutionarily-conserved cellular process represents another adaptive response to cellular stress. During autophagy cytosolic material, including organelles, proteins, and foreign pathogens, are sequestered into membrane-bound vesicles termed autophagosomes, and then delivered to the lysosome for degradation. Through recycling of cellular biochemicals, autophagy provides a mechanism for adaptation to starvation. Recent research has uncovered selective autophagic pathways that target distinct cargoes to autophagosomes, including mechanisms for the clearance of aggregated protein, and for the removal of dysfunctional mitochondria (mitophagy). Autophagy can be induced by multiple forms of chemical and physical stress, including endoplasmic reticulum stress and oxidative stress, and plays an integral role in the mammalian stress response. Understanding of the interaction and co-regulation of autophagy with other stress-inducible systems will be useful in the design and implementation of therapeutics targeting this pathway.
Linear polyubiquitin is processed at LRLRGG sequences by deubiquitinating enzymes to make free monomeric ubiquitin. This LRLRGG ubiquitin-like motif is found in a limited number of mammalian non-ubiquitin proteins, including the MAP3K Apoptosis Signal-Regulating Kinase-1 (ASK1), which activates MAPK signaling pathways. The c-terminus of ASK1 binds to the 19S cap of the proteasome allowing ASK1 to phosphorylate and inhibit proteasomal activity. We investigated whether the ubiquitin-like sequence in the c-terminus of ASK1 mediates its association with and inhibition of the proteasome. To test this we generated ASK1 with substitutions or deletions in this ubiquitin-like domain and examined the activation of cellular signaling and the association of ASK1 with the 19S cap of the proteasome. We show that ASK1 mutants have reduced association with the 19S cap of the proteasome, reduced capacity to inhibit the proteasome, and diminished ability to inhibit TNF-induced NF-κB activation. Mutant forms of ASK1 also had reduced capacity to activate JNK signaling, suggesting that the ubiquitin-like motif in ASK1 is also important for coordinating the balance between JNK and NF-κB signaling. Together these results demonstrate that the ubiquitin-like sequence of ASK1 is important for binding to and inhibition of the proteasome, and for the coordinated activation of cellular NF-κB and JNK signaling.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment and multiple pathological lesions. At the molecular level, AD is characterized by overt amyloid β (Aβ) production and tau hyper-phosphorylation. Hence, pharmacological agents that can attenuate Aβ accumulation and tau hyper-phosphorylation have potential promise for treatment of AD. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is believed to be one of such pharmacological agents. It is neuroprotective in neurodegenerative diseases and its primary action is thought to be via enhancement of autophagy, a biological process that not only facilitates the clearance of mutant proteins but also significantly reduces the build-up of toxic protein aggregates such as Aβ. Since rapamycin enhancement of autophagy has been associated with abrogation of AD pathological processes such as clearance of Aβ and neurofibrillary tangles (NTFs) as well as reduction of tau hyper-phosphorylation and improvement of cognition, rapamycin is emerging as a potential therapeutic compound for AD.
Protein oxidative modifications, also known as protein oxidation, are a major class of protein posttranslational modifications. They are caused by reactions between protein amino acid residues and reactive oxygen species (ROS) or reactive nitrogen species (RNS) and can be classified into two categories: irreversible modifications and reversible modifications. Protein oxidation has been often associated with functional decline of the target proteins, which are thought to contribute to normal aging and age-related pathogenesis. However, it has now been recognized that protein oxidative modifications can also play beneficial roles in disease and health. This review summarizes and highlights certain positive roles of protein oxidative modifications that have been documented in the literature. Covered oxidatively modified protein adducts include carbonylation, 3-nitrotyrosine, s-sulfenation, s-nitrosylation, s-glutathionylation, and disulfide formation. All of which have been widely analyzed in numerous experimental systems associated with redox stress conditions. The authors believe that selected protein targets, when modified in a reversible manner in prophylactic approaches such as preconditioning or ischemic tolerance, may provide potential promise in maintaining health and fighting disease.
Dihydrolipoamide dehydrogenase (DLDH) is a multifunctional oxidoreductase and is well known as an essential component of four mammalian mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched chain α-keto acid dehydrogenase, and the glycine cleavage system. However, existence of extracellular DLDH in mammals, if any, has not been clearly defined. The present article reports identification and biochemical characterization of serum DLDH. Proteomic analysis of rat serum using blue native polyacrylamide gel electrophoresis (BN-PAGE) and mass spectrometry peptide sequencing led to generation of 6 tryptic peptides in one band that matched to mitochondrial DLDH, indicating the existence of DLDH in rat serum. Measurement of enzymatic activity also indicated the existence of DLDH in human and mouse serum. Further biochemical analysis of rat serum DLDH revealed that this enzyme lacked diaphorase activity and could not be detected on Western blots probed with antibodies that recognized mitochondrial DLDH. Moreover, both ammonium sulfate fractioning and gel filtration of serum samples rendered a great loss in DLDH activity, indicating that the enzyme activity of this serum protein, unlike that of mitochondrial DLDH, is very labile. When DTT was supplemented in the buffer used for gel filtration, DLDH activity was found to be largely preserved; indicating that serum DLDH is susceptible to air-implicated inactivation. Results of the present study indicate that serum DLDH differs from mitochondrial DLDH in that it is a very labile enzyme.
