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Glyoxalase-1 (GLO-1) is the key enzyme in aldehyde defence in cancer cells. We here evaluated the prognostic impact and association with clinico-pathological parameters and relapse-free as well as overall survival in tumor samples from 187 breast cancer patients. The determined GLO1-immunoreactive score (GLO1-IRS) did not correlate with parameters such as grading, size, hormone receptors or ki67. However, an association of GLO1-IRS with the advanced glycation end product N ε- (carboxymethyl)lysine (p = 0.07) and HER2 (p = 0.06), and a strong correlation with VEGF (p = 0.008) was found. In survival analysis, no significant impact of GLO-1 IRS could be deduced for all patients. However, GLO1-IRS correlated with treatment by radiotherapy (p = 0.008) and high GLO1-IRS predicted a shorter relapse free survival after radiotherapy (log-rank p = 0.067). METABRIC- and TCGA expression-data were analyzed for correlation of regulatory genes of the NF-κB-pathway (RELA, RELB, IRAK1), the oxidative-stress associated transcription factor nrf2 (NFE2L2), the receptor for AGEs (AGER, RAGE) as well as enzymes associated with aldehyde defense. Here, RELA, RELB and NFE2L2 correlated significantly with GLO1 expression, but there were conflicting results between the two data sources. In conclusion, GLO1 was highly expressed in cancer cells, correlated surprisingly weak with survival, but we could show a positive association with the AGE CML as well as VEGF. Gene expression data suggest a regulation of GLO-1 mRNA via both, inflammation (NF-kB) and oxidative stress (NFE2L2) in tumors., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Objective: Sleep apnea has been associated with anxiety, but the mechanisms of the sleep apnea-anxiety relationship are unresolved. Sleep apnea causes oxidative stress, which might enhance anxiety-like behavior in rodents. To clarify the apnea-anxiety connection, we tested the effect of intermittent hypoxia, a model of sleep apnea, on the anxiety behavior of mice., Methods: The rodents were exposed daily to 480 one-minute cycles of intermittent hypoxia to a nadir of 7±1% inspiratory oxygen fraction or to a sham procedure with room air. After 7 days, the mice from both groups were placed in an elevated plus maze and were video recorded for 10 min to allow analysis of latency, frequency, and duration in open and closed arms. Glyoxalase-1 (Glo1) and glutathione reductase-1 (GR1) were measured in the cerebral cortex, hippocampus, and striatum by Western blotting., Results: Compared to controls, the intermittent hypoxia group displayed less anxiety-like behavior, perceived by a statistically significant increase in the number of entries and total time spent in open arms. A higher expression of GR1 in the cortex was also observed., Conclusion: The lack of a clear anxiety response as an outcome of intermittent hypoxia exposure suggests the existence of additional layers in the anxiety mechanism in sleep apnea, possibly represented by sleepiness and irreversible neuronal damage.

Background: Glyoxalase 1 is a key enzyme in the detoxification of reactive metabolites such as methylglyoxal and induced Glyoxalase 1 expression has been demonstrated for several human malignancies. However, the regulation and clinical relevance of Glyoxalase 1 in the context of head and neck squamous cell carcinoma has not been addressed so far., Methods: Argpyrimidine modification as a surrogate for methylglyoxal accumulation and Glyoxalase 1 expression in tumor cells was assessed by immunohistochemical staining of tissue microarrays with specimens from oropharyngeal squamous cell carcinoma patients (n = 154). Prognostic values of distinct Glyoxalase 1 staining patterns were demonstrated by Kaplan-Meier, univariate and multivariate Cox proportional hazard model analysis. The impact of exogenous methylglyoxal or a Glyoxalase 1 inhibitor on the viability of two established tumor cell lines was monitored by a colony-forming assay in vitro., Results: Glyoxalase 1 expression in tumor cells of oropharyngeal squamous cell carcinoma patients was positively correlated with the presence of Argpyrimidine modification and administration of exogenous methylglyoxal induced Glyoxalase 1 protein levels in FaDu and Cal27 cells in vitro. Cal27 cells with lower basal and methylglyoxal-induced Glyoxalase 1 expression were more sensitive to the cytotoxic effect at high methylgyoxal concentrations and both cell lines showed a decrease in colony formation with increasing amounts of a Glyoxalase 1 inhibitor. A high and nuclear Glyoxalase 1 staining was significantly correlated with shorter progression-free and disease-specific survival, and served as an independent risk factor for an unfavorable prognosis of oropharyngeal squamous cell carcinoma patients., Conclusions: Induced Glyoxalase 1 expression is a common feature in the pathogenesis of oropharyngeal squamous cell carcinoma and most likely represents an adaptive response to the accumulation of cytotoxic metabolites. Oropharyngeal squamous cell carcinoma patients with a high and nuclear Glyoxalase 1 staining pattern have a high risk for treatment failure, but might benefit from pharmacological targeting Glyoxalase 1 activity.

Cardiac arrest (CA) and cardiocerebral resuscitation (CCR)-induced ischemia-reperfusion imposes oxidative and carbonyl stress that injures the brain. The ischemic shift to anaerobic glycolysis, combined with oxyradical inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), provokes excessive formation of the powerful glycating agent, methylglyoxal. The glyoxalase (GLO) system, comprising the enzymes glyoxalase 1 (GLO1) and GLO2, utilizes reduced glutathione (GSH) supplied by glutathione reductase (GR) to detoxify methylglyoxal resulting in reduced protein glycation. Pyruvate, a natural antioxidant that augments GSH redox status, could sustain the GLO system in the face of ischemia-reperfusion. This study assessed the impact of CA-CCR on the cerebral GLO system and pyruvate's ability to preserve this neuroprotective system following CA. Domestic swine were subjected to 10 min CA, 4 min closed-chest CCR, defibrillation and 4 h recovery, or to a non-CA sham protocol. Sodium pyruvate or NaCl control was infused (0.1 mmol/kg/min, intravenous) throughout CCR and the first 60 min recovery. Protein glycation, GLO1 content, and activities of GLO1, GR, and GAPDH were analyzed in frontal cortex biopsied at 4 h recovery. CA-CCR produced marked protein glycation which was attenuated by pyruvate treatment. GLO1, GR, and GAPDH activities fell by 86, 55, and 30%, respectively, after CA-CCR with NaCl infusion. Pyruvate prevented inactivation of all three enzymes. CA-CCR sharply lowered GLO1 monomer content with commensurate formation of higher molecular weight immunoreactivity; pyruvate preserved GLO1 monomers. Thus, ischemia-reperfusion imposed by CA-CCR disabled the brain's antiglycation defenses. Pyruvate preserved these enzyme systems that protect the brain from glycation stress. Impact statement Recent studies have demonstrated a pivotal role of protein glycation in brain injury. Methylglyoxal, a by-product of glycolysis and a powerful glycating agent in brain, is detoxified by the glutathione-catalyzed glyoxalase (GLO) system, but the impact of cardiac arrest (CA) and cardiocerebral resuscitation (CCR) on the brain's antiglycation defenses is unknown. This study in a swine model of CA and CCR demonstrated for the first time that the intense cerebral ischemia-reperfusion imposed by CA-resuscitation disabled glyoxalase-1 and glutathione reductase (GR), the source of glutathione for methylglyoxal detoxification. Moreover, intravenous administration of pyruvate, a redox-active intermediary metabolite and antioxidant in brain, prevented inactivation of glyoxalase-1 and GR and blunted protein glycation in cerebral cortex. These findings in a large mammal are first evidence of GLO inactivation and the resultant cerebral protein glycation after CA-resuscitation, and identify novel actions of pyruvate to minimize protein glycation in postischemic brain.

Background: High concentrations of methylglyoxal (MGO) cause cytotoxiticy via formation of advanced glycation endproducts (AGEs) and inflammation. MGO is detoxificated enzymatically by glyoxalase-I (Glo-I). The aim of this study was to analyze the role of Glo-I during the development of cirrhosis., Methods: In primary hepatocytes, hepatic stellate cells (pHSC) and sinusoidal endothelial cells (pLSEC) from rats with early (CCl4 8wk) and advanced cirrhosis (CCl4 12wk) expression and activity of Glo-I were determined and compared to control. LPS stimulation (24h; 100ng/ml) of HSC was conducted in absence or presence of the partial Glo-I inhibitor ethyl pyruvate (EP) and the specific Glo-I inhibitor BrBzGSHCp2. MGO, inflammatory and fibrotic markers were measured by ELISA and Western blot. Additional rats were treated with CCl4 ± EP 40mg/kg b.w. i.p. from wk 8-12 and analyzed with sirius red staining and Western blot., Results: Expression of Glo-I was significantly reduced in cirrhosis in whole liver and primary liver cells accompanied by elevated levels of MGO. Activity of Glo-I was reduced in cirrhotic pHSC and pLSEC. LPS induced increases of TNF-α, Nrf2, collagen-I, α-SMA, NF-kB and pERK of HSC were blunted by EP and BrBzGSHCp2. Treatment with EP during development of cirrhosis significantly decreased the amount of fibrosis (12wk CCl4: 33.3±7.3%; EP wk 8-12: 20.7±6.2%; p<0.001) as well as levels of α-SMA, TGF-β and NF-κB in vivo., Conclusions: Our results show the importance of Glo-I as major detoxifying enzyme for MGO in cirrhosis. The reduced expression of Glo-I in cirrhosis demonstrates a possible explanation for increased inflammatory injury and suggests a "vicious circle" in liver disease. Blunting of the Glo-I activity decrease the amount of fibrosis in established cirrhosis and constitutes a novel target for antifibrotic therapy.

Objective: Glyoxalase 1 (GLO1) is ubiquitously expressed in the cytosol of the cell and is the major opponent against the reactive metabolite methylglyoxal, which is involved in the development of atherosclerosis. Nondiabetic individuals with an increased hemoglobin A1c (HbA1c) level are at higher risk for development of cardiovascular diseases. As such, this study investigated whether there was an association between reduced GLO1 activity in atherosclerotic lesions of nondiabetic patients with an increased HbA1c level., Methods: HbA1c level was determined in venous blood of patients with carotid artery disease. Protein level of GLO1 was measured in endarterectomy-derived carotid artery plaques by Western blotting. Activity was measured by spectrophotometric assay in the plaques as well as in the erythrocytes; GLO1 activity in erythrocytes was compared with that in a cohort of healthy individuals (n = 15; 33% men; average age, 60 years)., Results: There were 36 patients with carotid artery disease (69% men; average age, 69 years) included in this study and divided into two equal groups: group I, HbA1c < 5.7% (<39 mmol/mol); and group II, 5.7% ≤ HbA1c < 6.5% (39 mmol/mol ≤ HbA1c < 48 mmol/mol). GLO1 activity in carotid plaques was reduced by 29% in group II compared with group I (P = .048), whereas protein expression was unchanged (P = .25). Analysis of GLO1 activity in erythrocytes revealed no difference between the groups (P = .36) or in comparison to healthy controls (P = .15). Examination of clinical parameters showed an increased amount of patients with concomitant peripheral arterial disease in group II (44% vs 10%; P = .020)., Conclusions: Reduction of GLO1 activity in atherosclerotic lesions of nondiabetic patients with increased HbA1c is associated with a functional involvement of this protective enzyme in atherogenesis. Systemic GLO1 activity seems to be independent of both HbA1c and localized atherosclerosis as it was unchanged between group I and group II as well as compared with healthy controls, respectively., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Increased expression of receptor for advanced glycation end products (RAGE) in adipose tissue has been associated with inflammation, adipocyte hypertrophy, and impaired insulin signal. Epicardial adipose tissue (EAT), a visceral fat surrounding the myocardium, is potentially involved in the onset/progression of coronary artery disease (CAD). To date, the role of RAGE in EAT has not been explored much. We examined whether the RAGE expression in EAT was associated with EAT adiposity and metabolic dysfunctions normally found in CAD patients. EAT samples were obtained from 33 patients undergoing open-heart surgery. EAT expression of RAGE, GLUT4, adiponenctin, GLO1, HMGB1, TLR-4, and MyD88 was analyzed by microarray. EAT thickness was quantified by echocardiography. Anthropometric measures and clinical parameters were taken. BMI, HOMA-IR, and LAP indices were calculated. With increasing RAGE expression in EAT we observed increases in EAT thickness, reduced expression of GLUT4, adiponectin, and GLO1, and elevations of HMGB1, TLR-4, and MyD88. There were significant correlations between RAGE and EAT thickness and between RAGE and the genes. LAP was higher in patients with increased RAGE expression. Our data suggest that in CAD patients RAGE may be involved in promoting EAT adiposity and metabolic dysfunction, such as impaired insulin signaling.

Glyoxalase I (GLO1) is a methylglyoxal detoxification enzyme being implicated in the progression of multiple malignancies. However, currently, the role of GLO1 in human nonmelanoma skin tumors remains unclear. To explore the expression of GLO1 in cutaneous neoplasms and its role in the pathogenesis of skin cancers, we determined the GLO1 expression in multiple subtypes of cutaneous neoplasms and cell lines harboring different tumorigenicity. Also, the GLO1 siRNA transfection was performed in squamous cell carcinoma (SCC)-13 cells or SCC in the xenograft model. The results show that GLO1 was overexpressed by SCC, basal cell carcinoma, and verrucous carcinoma but weakly expressed by several benign neoplasms. Human papilloma virus 16 E6/E7-transfected keratinocytes expressed more GLO1 than did normal keratinocytes, although both of them had lower levels of GLO1 than SCC-13 cells. Moreover, the knockdown of GLO1 by siRNA was related to enhanced apoptosis of SCC-13 cells in the presence of tumor necrosis factor-related apoptosis-inducing ligand and inhibited cell invasion and migration, which was mirrored by the suppressed growth of SCC xenografts in mice. Finally, the GLO1 regulation of SCC-13 cells might be relevant to methylglyoxal-induced p53 translocation. Therefore, GLO1 is prevailingly expressed in cutaneous neoplasms of higher malignancy and contributes to the progression of SCC.

Lactoylglutathione lyase (GLO1), a ubiquitously expressed methylglyoxal (MG) detoxification enzyme, is implicated in the progression of various human malignant diseases. However, the role of GLO1 in the development or progression of murine fibrosarcoma is still unclear. We performed proteomic analysis to identify differences in the intracellular proteins of the regressive tumor cell line QR-32 and the inflammatory cell-promoting progressive tumor cell line QRsP-11 of murine fibrosarcoma by 2DE combined with MS. Seven upregulated proteins were identified in QRsP-11 compared to QR-32 cells, namely, GLO1, annexin A1, adenylate kinase isoenzyme 1, transcription factor BTF3, myosin light polypeptide 6, low molecular weight phosphotyrosine protein phosphatase and nucleoside diphosphate kinase B. Heat shock protein beta-1 (HspB1), a methylglyoxal-adducted protein, is concomitantly over-expressed in QRsP-11 as compared to QR-32 cells. We also found out that GLO1 is translocated into the nucleus to a higher extent in QRsP-11 compared to QR-32 cells, which can be reversed by using a MEK inhibitor (U0126). Moreover, U0126 and GLO1 siRNA can inhibit cell proliferation and migration in QRsP-11 cells. Our data suggest that overexpression and nuclear translocation of GLO1 might be associated with tumor progression in murine fibrosarcoma., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We examined whether or not grape powder treatment ameliorates oxidative stress-induced anxiety-like behavior, memory impairment, and hypertension in rats. Oxidative stress in Sprague-Dawley rats was produced by using L-buthionine-(S,R)-sulfoximine (BSO). Four groups of rats were used: 1) control (C; injected with vehicle and provided with tap water), 2) grape powder-treated (GP; injected with vehicle and provided for 3 wk with 15 g/L grape powder dissolved in tap water), 3) BSO-treated [injected with BSO (300 mg/kg body weight), i.p. for 7 d and provided with tap water], and 4) BSO plus grape powder-treated (GP+BSO; injected with BSO and provided with grape powder-treated tap water). Anxiety-like behavior was significantly greater in BSO rats compared with C or GP rats (P < 0.05). Grape powder attenuated BSO-induced anxiety-like behavior in GP+BSO rats. BSO rats made significantly more errors in both short- and long-term memory tests compared with C or GP rats (P < 0.05), which was prevented in GP+BSO rats. Systolic and diastolic blood pressure was significantly greater in BSO rats compared with C or GP rats (P < 0.05), whereas grape powder prevented high blood pressure in GP+BSO rats. Furthermore, brain extracellular signal-regulated kinase-1/2 (ERK-1/2) was activated (P < 0.05), whereas levels of glyoxalase-1 (GLO-1), glutathione reductase-1 (GSR-1), calcium/calmodulin-dependent protein kinase type IV (CAMK-IV), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) were significantly less (P < 0.05) in BSO but not in GP+BSO rats compared with C or GP rats. We suggest that by regulating brain ERK-1/2, GLO-1, GSR-1, CAMK-IV, CREB, and BDNF levels, grape powder prevents oxidative stress-induced anxiety, memory impairment, and hypertension in rats.

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [(3)H] B[a]P, but increased the cellular discharge of [(3)H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500μM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

Autism is a neurodevelopmental disability characterized by deficits in verbal communications, impairments in social interactions, and repetitive behaviors. Several studies have indicated strong involvement of multigenic components in the etiology of autism. Linkage analyses and candidate gene search approaches so far have not identified any reliable susceptibility genes. We are using a proteomic approach to identify protein abnormalities due to aberrant gene expression in autopsied autism brains. In four of eight autism brains, we have found an increase in polarity (more acidic) of glyoxalase I (Glo1) by two-dimensional gel electrophoresis. To identify the molecular change resulting in the shift of Glo1 polarity, we undertook sequencing of GLO1 gene. Direct sequencing of GLO1 gene/mRNA in these brains, has identified a single nucleotide polymorphism (SNP), C419A. The SNP causes an Ala111Glu change in the protein sequence. Population genetics of GLO1 C419A SNP studied in autism (71 samples) and normal and neurological controls (49 samples) showed significantly higher frequency for the A419 (allele frequency 0.6 in autism and 0.4 in controls, one-tailed Fisher's test P < 0.0079). Biochemical measurements have revealed a 38% decrease in Glo1 enzyme activity in autism brains (one-tailed t-test P < 0.026). Western blot analysis has also shown accumulation of advanced glycation end products (AGE's) in autism brains. These data suggest that homozygosity for A419 GLO1 resulting in Glu111 is a predisposing factor in the etiology of autism., ((c) 2004 Wiley-Liss, Inc.)

Cereal proteins are known to cause allergic reactions such as Baker's asthma and severe atopic dermatitis to certain populations. In rice allergy, proteins with molecular masses of 14-16, 26, 33, and 56 kDa have been demonstrated to be potentially allergenic. In this study, to identify and characterize the 33-kDa allergen, designated Glb33, this protein was first purified to homogeneity, and its cDNA clone was isolated. When expressed in Escherichia coli, the recombinant Glb33 was shown to be as reactive as the native Glb33 with mouse IgG and patients' IgE antibodies to Glb33. The Glb33 cDNA coded for a protein of 291 amino acids with two 120-amino acid residue repeats, and the amino acid sequence showed similarity to glyoxalase I from various organisms, including human, plant, yeast, and bacterium. As expected, both native Glb33 purified from rice seeds and the recombinant protein had glyoxalase I activity that catalyzes condensation of methylglyoxal and glutathione into S-lactoylglutathione. However, Glb33 had a higher sequence identity to the bacterial glyoxalase I rather than to known plant and yeast enzymes. Both the Glb33 transcript and the protein were detected not only in maturing seeds of rice but also in its stem and leaf. Taken all together, the rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds.

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

Although the glyoxalase system was discovered in 1913, its function in the biological network is still a subject of debate. An attractive theory on its role was described by Albert Szent-Györgyi in the 1960s. From a bird's eye view, the promine/retine concept of Szent-Györgyi seems to give a plausible role for this ubiquitous enzyme system, but on going into detail, it obviously suffers from several uncertainties which have not been discussed until now. Here, a critical overview of the theory is presented by taking the pros and cons into account. It looks as though more data object to the theory than give support to it; and the search for anticancer medicines stimulated by the theory has not resulted in a new way of treatment of tumors, either. Hence, it is feared that the theory suggested for the biological role of glyoxalase pathway cannot be accepted, as it is.

The steady state expression of glutathione S-transferases (GSTs) at both the protein and mRNA level is reported for the 60 tumor cell lines that are used for the National Cancer Institute Drug Screening Program. Individual GST isozymes were separated, identified, and quantified (with reverse-phase calibration curves) through a novel high performance liquid chromatographic procedure. GSTP1 was the predominant isozyme and was found at quantifiable levels in all but two of the cell lines. This isozyme ranged from 0.03% to 2.7% of the total cytosolic protein. For the mu family, 90% of the lines had GSTM2, 68% had GSTM3, but only 28% were positive for the M1 phenotype. The M1 proportion is lower than would be expected from the standard M1 null phenotype for human populations. Isozymes of the alpha family were detected only at very low levels in 35% of the lines. Significant quantitative correlations among enzyme activity, total enzyme protein, and mRNA were shown for GSTP1. However, such relationships were not apparent for the mu or alpha families. Levels of glutathione (GSH), and the transcript levels of other enzymes involved in GSH homeostasis were determined. gamma-Glutamyl cysteine synthetase (gamma-GCS) was present in all cell lines, but did not correlate with levels of intracellular GSH. Glyoxalase-I and gamma-glutamyl transpeptidase, both involved in GSH salvage, were found in 100% and 70% of the cell lines, respectively. Using a pattern-matching computer program, COMPARE, we compared and correlated the arrays of mRNA and protein levels with the pattern of chemosensitivity or chemoresistance of the 60 cell lines with 175 agents constituting a standard agent database. This database is composed of compounds to which a putative mechanism of action has been assigned. Although Pearson correlation coefficients relating the target and drug patterns were generally modest, when the patterns for the enzyme protein and mRNA levels for GST pi were correlated to drug sensitivity patterns, the list of 30 agents most closely matching (for which P < 0.05) was enriched with alkylating agents. gamma-GCS also showed an enrichment of alkylating agents in the COMPARE correlations, indicating that high levels of gamma-GCS may be an important determinant of resistance. In contrast, none of the other enzymes or GSH had patterns of expression that resulted in an obvious correlation to the sensitivity or resistance of alkylating agents.

We have isolated two kinds of cDNA clones encoding glyoxalase I from a human fibrosarcoma HT-1080 cDNA library. One of them is identical to the glyoxalase I cDNA isolated by us, and the other encodes a protein in which alanine at position 111 of the reported sequence for glyoxalase I is replaced by glutamic acid. When the two cDNAs were co-translated in vitro, three bands representing two homodimers and one heterodimer appeared on native polyacrylamide gel electrophoresis, as observed for glyoxalase I purified from human erythrocytes or derived from a HT-1080 cell lysate. Escherichia coli cells carrying an expression vector of one of the novel glyoxalase I cDNAs showed glyoxalase I activity. These results reveal that two isoforms of human glyoxalase I showing different electrophoretic properties result from a change in one amino acid residue.

The activities of glyoxalase I and glyoxalase II and the concentration of methylglyoxal were determined in 26 human lenses. The activity of glyoxalase I (mean +/- S.D.) was 15.62 +/- 3.90 U (g wet weight)-1 and the activity of glyoxalase II was 0.189 +/- 0.087 U (g wet weight)-1 (n = 26). The concentration of methylglyoxal of the human lenses was 1.78 +/- 0.84 nmol (g wet weight)-1 (n = 26). There was a significant negative correlation of both the activity of glyoxalase I and the activity of glyoxalase II with subject age but no correlation of methylglyoxal concentration with subject age. The concentration of methylglyoxal in the lenses was approximately 20-fold higher than in blood samples from normal human subjects. Given the previously reported decrease in the concentration of reduced glutathione in the human lens with age, there is expected to be a marked decrease in in situ activity of glyoxalase I and concomitant susceptibility of human lens proteins to modification by methylglyoxal with age. The metabolism of methylglyoxal and the formation of methylglyoxal-modified proteins may be linked to the development of senile and diabetic cataract.

Glyoxalase-I (Gly-I) is part of the glyoxalase system which converts methylglyoxal to D-lactic acid via an S-D-lactoylglutathione intermediate. This glutathione (GSH)-binding protein was purified from human colon tumors and corresponding normal tissue. The GSH-affinity purified fraction from normal human colon tissue showed enzyme activity of 30.6 +/- 11.5 mumol/min per mg protein, with methylglyoxal as substrate. Corresponding fractions from carcinomas showed significantly elevated Gly-I activity of 54.5 +/- 15 mumol/min per mg protein. Polyclonal antibodies made against human Gly-I cross-reacted weakly with mouse liver Gly-I but not with yeast Gly-I. Isoelectric points of Gly-I from human, mouse and yeast were determined to be 4.6, 4.9 and 7.0, respectively, by horizontal IEF. Immunohistochemical analysis confirmed the increase of Gly-I in human colon carcinoma in 16 out of 21 samples when compared to corresponding normal tissue. The elevated levels of Gly-I in colon tumors may be an indicator of the enhanced proliferative status of the neoplastic condition.

Objective: To find the degree of HLA antigen sharing in couples with infertility of unknown etiology and compare it with that in couples with infertility of known etiology as well as couples with normal fertility., Subjects: Eleven couples with infertility of unknown etiology, 26 with infertility of known etiology, and 31 fertile couples were tested for HLA class I (A, B, C), class II (DR, MLC), and class III (Bf) antigens and GLO alleles. HLA lymphocytotoxic antibodies and anti-sperm antibodies in both partners were also searched., Results: Gene frequencies and the number of HLA antigens shared between the two members of the couple were similar in all groups. When "intra-couple" MLC reactivity was measured, no quantitative differences were found in the infertile group with unknown etiology, as compared to the group of known etiology: spouses in couples of either group usually reacted in both ways as expected for unrelated individuals" lymphocytes. Lymphocytotoxic antibody frequency did not differ between the nonfertile groups, and anti-sperm antibodies, found in ten patients in the group of known etiology, were not associated with any HLA antigen. In spite of that, GLO and all the HLA antigen classes described (I, II and III) were analyzed., Conclusions: Although the results are negative, these and others' (negative) results concerning couples with infertility of unknown etiology do not disprove that HLA has a role in fertilization, because HLA-related factors still unknown may exist. Exhaustive HLA available marker typing in international cooperative efforts may be needed to reach a significant number of carefully selected couples fully HLA typed to elucidate this problem.

Paired human teeth and fresh blood samples were analysed for the genetic markers ABO, PGM1, GLO-I, EsD, EAP, AK, ADA, 6-PGD, G-6-PD and CA-II. ABO blood groups were successfully determined from dentine, pulp and cement samples whereas only dental pulp could be typed for different isozymes. Studies on the persistence of these genetic markers in teeth stored at room temperature showed that ABO blood groups were the most stable, followed by PGM1 and ADA isozymes, whereas CA-II isozymes were the least stable.

Glutathione transferase, glyoxalase I and glyoxalase II activities were not evenly distributed among the major helminth groups. Intestinal cestodes and digeneans had higher glutathione transferase activity than parasitic nematodes. High glyoxalase II activity was found in cestodes and digeneans but no glyoxalase I was detectable. Glyoxalase I and II were both detected in nematodes. These results are discussed in relation to the enzymes' suggested role in protection against secondary lipid peroxidation products.

Possibility of combined determination of phenotypes of three enzyme system (glyoxylase-1, phosphoglucomutase and 6-phosphogluconate dehydrogenase) in the apparatus with two vertical blocks of polyacrilamide gel (PAAG) was shown. The method is based on the principle of combination of differently composed blocks of PAAG and single universal electrode buffer. High productivity and economy of the method equally with good quality of resulting phoregrams make this method useful for medicolegal practical work.

Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.

A technique is described for the typing of glyoxalase I (GLO I) and the subtyping of phosphoglucomutase-1 (PGM-1) from the root sheath cells of a single forcibly removed hair. This procedure does not require sample preparation and does not alter the morphological characteristics of the hair. The combined discrimination probability (DP) of the two markers taken together is 0.90 for whites and 0.89 for blacks. GLO I can be typed after four weeks, and PGM-1 can be typed after eight to fifteen weeks in hairs maintained at room temperature. Hairs mounted with Permount showed loss of enzyme activity and loss of band sharpness.

The grouping of blood/saliva samples from a male so as to predict his semen groups is only justified if there is a strict correlation between the groupings in these body fluids. This correlation has been examined in the ABO, phosphoglucomutase (PGM1) and glyoxalase I (GLO) grouping systems in blood and semen samples collected from more than 250 individuals. Though no results proved inconsistent with this correlation, a number of semen gave inconclusive grouping results. Reasons for this are discussed as well as the relevance of the results to semen stain analysis. Semen amylase activities are also reported.

Insulin-dependent diabetics are not equal as concerns vascular complications. Many factors have been incriminated. The object of this study was to determine if a genetic factor protected the insulin-dependent diabetic from vascular complications. The population included 31 unrelated, white insulin-dependent diabetic subjects (18 females and 13 males), with diabetes of over 20 years' duration (21 to 43 years, m +/- SD 27.82 +/- 6.03 years; age at diagnosis: 2 to 57 years, m +/- SD 22.7 +/- 15.19 years). The absence of vascular lesions was checked for by the following investigations: visual acuity, fundoscopic examination, retinal fluorescein angiography (Canon F 60 Z), systolic pressure on the thigh and ankle, Doppler velocimetry, plethysmography of the lower limbs, serum creatinine level, urinary protein. The study of the HLA A, B, DR specificities was carried out using the Tersaki microlymphocytotoxicity assay, those of C4 by high voltage gel electrophoresis followed by hemolytic detection, those of B1 using Alper's method and those of glyoxalase by gel electrophoresis followed by a glutathione redox reaction in order to test for a marker for a possible protective genetic factor against complications. The antigen frequencies found were compared with: 1) Those in the general Caucasian reference population (9th Histocompatibility Workshop, 1984). The study group presented the HLA characteristics known to occur in insulin-dependent diabetes: increase in A30, B8, B18, D6, DR3, DR4, BfF1 and decrease in A3, B7, DR2. Furthermore an increase in the frequency of DRw53, DQw2 and DQw3 alleles was noted.(ABSTRACT TRUNCATED AT 250 WORDS)

A consanguineous family has been typed for HLA-A, B, C, D, DR and GLO, Bf, C2 and C4 and other red cell markers. The results indicate that an expected Dw7 homozygous sibling is a "Dw1"/Dw7 recombinant, probably derived from a crossing over between DR and D on the paternal haplotypes. The anomalous typings by Dw1 HTCs of the recombinant haplotype are best explained by the likely presence of an additional Lad polymorphism which could be a serologically detectable second epitope on the same molecule as the HLA-D determinant or in the form of two linked chains, one encoded by HLA-D and one by another Lad gene in the HLA haplotype.

The genetic structure of three subpopulations of the Naikpod tribe of Andhra Pradesh, India, was examined by studying three blood group, six red cell enzyme, and five protein systems and phenylthiocarbamide taste sensitivity. The gene frequency data of 15 loci are compared among the subpopulations as well as with those reported for other population groups from India. The analysis of gene diversity revealed that the gene differentiation among the subpopulations relative to total population is only 0.02, indicating that the genetic differentiation between subpopulations is very small compared with that within them. This is corroborated by the small genetic distances found among them. The effect of differentiation of microgeographical and breeding isolation on gene diversity and genetic differentiation among the three subpopulations is apparently low.

Glyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1,400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36,000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37 degrees C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4,5-dioxovalerate with Km values of 1.25 and 0.87 mM, respectively. The activity of the enzyme was completely inhibited by Zn2+ at 0.5 mM. An equimolar amount of EDTA (0.5 mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1 = 1.3 mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5 mM.

A technique was developed to type the glyoxalase I (GLO I) isoenzymes using a mixed agarose/starch gel. Over six hundred blood samples from Caucasoid people living in separate regions of South Australia were examined and the results compared with other Caucasoid population surveys. Paired blood and semen samples were also tested and the limitations of the technique with regard to blood and semen stains analysis was evaluated.

HLA-A,B,C and DR antigens were tested in 75 Cape Coloured systemic lupus erythematosus (SLE) patients, and the GLO I and Bf markers in 51. The patients with HLA-DR2 had a relative risk significantly greater than one (p=0.0005). Twenty-two (29%) patients had only one detectable DR antigen. Of these, 11 (50%) were found to have DR2 only. The HLA-DR7 antigen was associated with severe disease (p less than 0.02). Bf and GLO I markers were not associated with SLE.

Bacteria present in the human gut may produce methylglyoxal--a cytotoxic substance in mammals. This was investigated by studying the activity of methylglyoxal synthase, which produces methylglyoxal from dihydroxyacetone phosphate, and methylglyoxal concentration in growth medium of various bacteria isolated from human faeces. Facultative and strictly anaerobic bacteria isolated from faeces were able to produce methylglyoxal in both defined and complex media. Proteus spp. produced large amounts of methylglyoxal and had the greatest methylglyoxal synthase activity. Supplementing defined medium for facultative anaerobes with glucose 1% w/v did not significantly alter enzyme activity or methylglyoxal production. Inclusion of short chain fatty acids or bile acids in the medium reduced methylglyoxal synthase activity and methylglyoxal production by Proteus spp. None of the organisms examined had amine oxidase activity which could have contributed to methylglyoxal production from aminoacetone.