Search

Background: Diabetic neuropathy (DN) is the least recognized complication of diabetes mellitus and may start early in the course of the disease. Aldose reductase (AKR1B1) gene promoter Z-2/Z-2 polymorphism increases the expression of AKR1B1 enzyme and may contribute to DN., Subjects: We evaluated 108 Type 1 diabetes (T1D) children and adolescents (mean ± SD age: 13.5 ± 3.46 years, disease duration: 5.3 ± 3.4 years) and 150 healthy controls (age: 11.9 ± 2.7 years)., Methods: In both groups, pupillary dilation (PD) in darkness, postural blood pressure test (PBPT), and vibration sensation thresholds (VST) in upper and lower limbs were estimated as indices of autonomic and peripheral neuropathy, respectively. Nerve conduction studies (NCS) were performed in patients as peripheral neuropathy index. The polymorphisms of AKR1B1 gene were evaluated using microsatellite (AC)n sequence Z., Results: PBPT, PD, and VST impairments were more frequent in patient group compared with controls, while 38.6% of patients exhibited NCS abnormality. Gender, age, pubertal status, height, body mass index, diabetes duration, HbA1c, and anti-GAD titers were associated with neuropathy indices in patients. There was a strong correlation between PD and NCS in patients, while homozygous patients for Z-2 AKR1B1 gene polymorphism had higher prevalence of abnormal NCS (83.3% vs. 34.6%), PD (62.5% vs. 31.5%), and PBPT values compared with heterozygous or negative patients. Homozygous AKR1B1 status predicted PD, NCS, and PBPT variance, while PD, VST, NCS, and PBPT parameters accurately discriminated homozygous AKR1B1 patients., Conclusions: Impaired indices of peripheral and autonomic DN were present in a significant proportion of young T1D patients. PD, VST, NCS, and PBPT parameters were simultaneously associated with homozygous state of AKR1B1 Z-2 gene polymorphism, implicating polyol metabolism with both autonomic and peripheral neuropathies., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: To investigate if remote ischemic preconditioning (RIPC) can offer any renoprotective value by counteracting the deleterious effect of partial nephrectomy (PN) under warm ischemia on renal function., Methods: Four groups, each with 5 Wistar albino rats, were constructed; RIPC + PN, PN, RIPC and sham. Right nephrectomy was performed to constitute a solitary kidney model. RIPC denoted sequential clamping/declamping of the femoral artery/vein complex. PN was performed under warm-ischemia following RIPC. Blood samples were collected on multiple occasions until euthanasia on day 7. Immunoassays were conducted to measure the serum and tissues levels of kidney injury markers. Kidneys were examined histologically and morphometric analyzes were performed using digital scanning., Results: IL-33 levels did not differ significantly between the groups. Serum levels of KIM-1, NGAL, and aldose reductase in RIPC + PN, PN and RIPC groups were significantly lower than that of sham group. Tissue biomarker levels were similar across groups. The observed trend in mean necrosis area of PN group was higher than that of RIPC + PN group (p > 0.05). The transitional zone between necrosis and healthy tissue showed a trend towards increasing width in the rats subjected to RIPC before PN vs. those who underwent PN without RIPC (p > 0.05)., Conclusion: RIPC failed to counteract the renal functional consequences of PN under warm ischemia in a solitary kidney animal model. The supportive but marginal histological findings in favor of RIPC's renoprotective potential were not supplemented with the changes in serum and tissue biomarker levels., (© 2021. The Author(s).)

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins β actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Cystic pancreatic tumors account for 10% of cystic lesions in the pancreas. Evaluation focuses on identifying lesions that require surgical resection due to actual or potential malignancy. Cystic tumors with malignant potential include mucinous cystic neoplasms (MCNs), intraductal papillary mucinous neoplasms (IPMNs), and cystic neuroendocrine tumors (NETs). The sensitivity of endoscopic fine needle aspiration (FNA) to diagnose such lesions is low, and a more accurate marker of malignant potential is needed. Aldo-keto reductase 1B10 (AKR1B10) was originally found in human hepatocellular carcinoma. Since then, it has been identified in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia. Because there is difficulty in determining the malignant potential of cystic pancreatic tumors, we set out to examine the expression of AKR1B10 in these lesions as a potential biomarker of malignancy. AKR1B10 expression was analyzed in cell blocks from FNAs and surgical resection specimens using immunohistochemistry. We examined MCN (n=28), IPMN (n=18), and cystic NET (n=20) as well as nonmucinous cysts including pseudocysts (n=13) and serous cystadenomas (n=16). AKR1B10 expression was seen in 45 of 46 (98%) mucinous lesions evaluated. Strong staining (2+-3+/60%-100% staining) was seen in 16 of 18 (89%) IPMNs and 25 of 28 (90%) MCNs. No staining was seen in the nonmucinous lesions (n=49). In conclusion, AKR1B10 is upregulated in mucinous cystic pancreatic tumors, and this staining can be accomplished in cytology FNA material, making AKR1B10 a promising biomarker of malignant potential. Most importantly, this application could impact the clinical management of these patients by determining the best candidates for surgical resection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The incidence of breast cancer in India is on the rise and is rapidly becoming the primary cancer in Indian women. The aldoketo reductase (AKR) family has more than 190 proteins including aldose reductase (AKR1B1) and aldose reductase like protein (AKR1B10). Apart from liver cancer, the status of AKR1B1 and AKR1B10 with respect to their expression and activity has not been reported in other human cancers. We studied the specific activity and expression of AKR1B1 and AKR1B10 in breast non tumor and tumor tissues and in the blood. Fresh post-surgical breast cancer and non-cancer tissues and blood were collected from the subjects who were admitted for surgical therapy. Malignant, benign and pre-surgical chemotherapy samples were evaluated by histopathology scoring. Expression of AKR1B1 and AKR1B10 was carried out by immunoblotting and immunohistochemistry (IHC) while specific activity was determined spectrophotometrically. The specific activity of AKR1B1 was significantly higher in red blood cells (RBC) in all three grades of primary surgical and post-chemotherapy samples. Specific activity of both AKR1B1 and AKR1B10 increased in tumor samples compared to their corresponding non tumor samples (primary surgical and post-chemotherapy). Immunoblotting and IHC data also indicated overexpression of AKR1B1 in all grades of tumors compared to their corresponding non tumor samples. There was no change in the specific activity of AKR1B1 in benign samples compared to all grades of tumor and non-tumors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The porcine corpus luteum (CL) displays delayed sensitivity to PGF-2α (luteolytic sensitivity, [LS]) until days 12 to 13 of cycle. The control of LS is unknown, but it is temporally associated with macrophage (which secrete tumor necrosis factor-α; TNF-α) infiltration into the CL. Other studies showed that TNF-α induces LS in vitro and that prostaglandins (PGs) may be involved in this mechanism. In experiment 1, PGF-2α and PGE secretion by luteal cells (LCs) was measured on days 4 to 14 of the estrous cycle, and the expression of PTGFS/AKR1B1 and PTGES/mPGES-1, determined by Western blot, before (day 7) vs after (day 13) the onset of LS. Results showed that the PGF-2α:PGE ratio increased significantly (P < 0.05) from day 4 to 13-14, and PTGFS/AKR1B1 and PTGES/mPGES-1 were significantly increased (P < 0.05) on day 13 (vs day 7). In experiment 2, LCs were collected from porcine CL at early (∼days 4-6) or mid (∼days 7-12) stages of the estrous cycle and cultured with 0, 0.1, 1, or 10 ng/mL TNF-α. Results showed that TNF-α significantly increased (P < 0.05) messenger RNA (mRNA) expression of cyclooxygenase (COX)-2 and mPGES-1 but not AKR1B1. TNF-α had no significant effects on AKR1B1 or mPGES protein abundance. TNF-α significantly increased (P < 0.05) PGE-2 but had no effect on PGF-2α secretion or on the PGF-2α:PGE2 ratio. In conclusion, although TNF-α increased COX2 and mPGES-1 mRNA, and PGE-2 secretion in vitro, it did not increase the PGF-2α:PGE2 ratio. Studies are currently directed toward exploring other pathways (eg, FP receptor signaling) by which TNF-α induces LS in the porcine CL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Electrophilic carbonyl compounds are highly cytotoxic and genotoxic. Aldo-keto reductase 1B10 (AKR1B10) is an enzyme catalyzing reduction of carbonyl compounds to less toxic alcoholic forms. This study presents novel evidence that AKR1B10 protects colon cells from DNA damage induced by electrophilic carbonyl compounds. AKR1B10 is specifically expressed in epithelial cells of the human colon, but this study found that AKR1B10 expression was lost or markedly diminished in colorectal cancer, precancerous tissues, and a notable portion of normal adjacent tissues (NAT). SiRNA-mediated silencing of AKR1B10 in colon cancer cells HCT-8 enhanced cytotoxicity of acrolein and HNE, whereas ectopic expression of AKR1B10 in colon cancer cells RKO prevented the host cells against carbonyl cytotoxicity. Furthermore, siRNA-mediated AKR1B10 silencing led to DNA breaks and activation of γ-H2AX protein, a marker of DNA double strand breaks, particularly in the exposure of HNE (10 μM). In the AKR1B10 silenced HCT-8 cells, hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency increased by 26.8 times at basal level and by 33.5 times in the presence of 10 μM HNE when compared to vector control cells. In these cells, the cyclic acrolein-deoxyguanosine adducts levels were increased by over 10 times. These findings were confirmed by pharmacological inhibition of AKR1B10 activity by Epalrestat. Taken together, these data suggest that AKR1B10 is a critical protein that protects host cells from DNA damage induced by electrophilic carbonyl compounds. AKR1B10 deficiency in the colon may be an important pathogenic factor in disease progression and carcinogenesis. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Diabetes is one of the major chronic diseases diagnosed worldwide with a common complication of diabetic nephropathy (DN). There are multiple possible mechanisms associated with DN. Aldose reductase (AR) and Toll-like receptor 4 (TLR4) may be involved in the occurrence and development of DN. Here, we describe the distribution of AR and TLR4 in cells and renal tissues of diabetic rats through a quantum dot (QD)-based immunofluorescence technique and conventional immunohistochemistry. As a new type of nanosized fluorophore, QDs have been recognized in imaging applications and have broad prospects in biomedical research. The results of the reported study demonstrate that both the AR and the TLR4 proteins were upregulated in the renal tissues of diabetic rats. Further, to explore the relationship between AR and TLR4 in the pathogenesis of DN, a dual-color immunofluorescent labeling technique based on QDs was applied, where the expressions of AR and TLR4 in the renal tissues of diabetic rats were simultaneously observed - for the first time, as far as we are aware. The optimized QD-based immunofluorescence technique has not only shown a satisfying sensitivity and specificity for the detection of biomarkers in cells and tissues, but also is a valuable supplement of immunohistochemistry. The QD-based multiplexed imaging technology provides a new insight into the mechanistic study of the correlation among biological factors as well as having potential applications in the diagnosis and treatment of diseases.

Objective: To investigate enzymatic reactive aldehyde-scavenging enzyme capacity together with lipid peroxidation as expression of oxidative stress in atherosclerotic plaques of cigarette smokers and nonsmokers., Methods: We have assessed specific enzymatic activities of class 1, 2, and 3 aldehyde dehydrogenase (ALDH1, ALDH2, and ALDH3, respectively), glutathione S-transferase (isozyme A4-4, GSTA4-4), and aldose reductase (AR), namely the major reactive aldehyde-scavenging enzymes, together with lipid peroxidation, i.e., fluorescent damage products of lipid peroxidation (FDPL), in carotid atherosclerotic plaques surgically removed from 17 cigarette smokers and 17 nonsmokers., Results: The enzymatic activities of ALDH1 plus ALDH2, ALDH3, GSTA4-4, and AR were significantly lower in the atherosclerotic plaques of smokers than in those of nonsmokers, while plaque FDPL levels were significantly higher in the smokers than in the nonsmokers. The amount of cigarette smoking was correlated inversely with the aforementioned plaque enzymatic activities and directly with plaque FDPL content. Plaque FDPL levels were inversely correlated with plaque enzymatic activities in smokers and nonsmokers. The degree of carotid atherosclerotic stenosis, as expression of atherosclerosis severity, was correlated inversely with plaque enzymatic activities and directly with plaque FDPL levels in smokers and nonsmokers; moreover, the degree of carotid stenosis was directly correlated with the amount of cigarette smoking., Conclusion: atherosclerotic lesions of cigarette smokers are endowed with a depressed enzymatic reactive aldehyde-scavenging capacity eventually favoring oxidative stress and the severity of atherosclerosis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Elucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour., Methods: Expression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry., Results: Expression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types., Conclusions: Preterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.

Purpose: The intracardiac synthesis of anthracycline alcohol metabolites (e.g., daunorubicinol) contributes to the pathogenesis of anthracycline-related cardiotoxicity. Cancer patients with Down syndrome (DS) are at increased risk for anthracycline-related cardiotoxicity. We profiled the expression of anthracycline metabolizing enzymes in hearts from donors with- and without- DS., Methods: Cardiac expression of CBR1, CBR3, AKR1A1, AKR1C3 and AKR7A2 was examined by quantitative real time PCR, quantitative immunoblotting, and enzyme activity assays using daunorubicin. The CBR1 polymorphism rs9024 was investigated by allelic discrimination with fluorescent probes. The contribution of CBRs/AKRs proteins to daunorubicin reductase activity was examined by multiple linear regression., Results: CBR1 was the most abundant transcript (average relative expression; DS: 81%, non-DS: 58%), and AKR7A2 was the most abundant protein (average relative expression; DS: 38%, non-DS: 35%). Positive associations between cardiac CBR1 protein levels and daunorubicin reductase activity were found for samples from donors with- and without- DS. Regression analysis suggests that sex, CBR1, AKR1A1, and AKR7A2 protein levels were significant contributors to cardiac daunorubicin reductase activity. CBR1 rs9024 genotype status impacts on cardiac CBR1 expression in non-DS hearts., Conclusions: CBR1, AKR1A1, and AKR7A2 protein levels point to be important determinants for predicting the synthesis of cardiotoxic daunorubicinol in heart.

Hepatocellular carcinoma (HCC) is one of the most common highly aggressive malignant tumors worldwide. Aldoketoreductase 1B10 (AKR1B10) was first isolated from HCC and further identified to be over-expressed in many cancers from various organs. AKR1B10 contributes to detoxification of xenobiotics by lipid peroxidation and metabolizes physiological substrates such as farnesal, retinal, and carbonyls. Metabolizing these lipid substrates plays a crucial role in promoting carcinogenesis. In the present study, immunohistochemical analysis was performed to determine the prevalence/pattern of AKR1B10 expression in HCC and its usefulness to differentiate benign liver lesions from HCC. Oncogenic function of AKR1B10 was examined in hepatocellular carcinoma cells in vitro using Western blotting and shRNA knockdown approaches, with emphasis on cell apoptosis and response to chemotherapy. Immunohistochemistry analysis revealed AKR1B10 was overexpressed in 97% (86/89) of hepatocellular carcinomas, with minimal to no expression in adjacent hepatic tissue, while hepatic adenomas and focal nodular hyperplasia did not exhibit expression of AKR1B10. shRNA-mediated silencing of AKR1B10 expression in hepatocellular carcinoma cells resulted in (1) increased cell apoptosis, (2) decreased colony formation and size, and (3) enhanced cytoreductive response following exposure to doxorubicin chemotherapy. Our findings provide first time evidence that AKR1B10 is a unique biomarker involved in hepatocellular carcinogenesis via modulation of proliferation, cell apoptosis and chemoresistance and is a potential promising biomarker to differentiate HCCs from benign hepatic lesions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background and Objectives: The aim of the study was to investigate the correlation between AKR1B10 expression and clinicopathological features of gastric cancer (GC)., Methods: Real-time polymerase chain reaction (RT-PCR) was performed to determine AKR1B10 mRNA expression. AKR1B10 protein levels were measured by immunohistochemistry., Results: RT-PCR analysis confirmed that AKR1B10 was significantly down-regulated in gastric cancer compared with paired, normal mucosa. Immunohistochemistry revealed that the percentage of AKR1B10-positive specimens was lower in gastric carcinoma compared with normal specimens. The frequency of AKR1B10-positive GC specimens was higher in patients with tumor size <5 cm, no lymph node metastasis, no distant metastasis and lower tumor stages The mean survival time for patients in the AKR1B10-positive group was significantly higher compared with the AKR1B1-negative group. The 5-year survival rate for the AKR1B10-positive group was also significantly higher than for the AKR1B1-negative group. Cox regression analysis revealed that AKR1B10 expression is an independent prognostic factor of GC., Conclusions: Expression of AKR1B10 in gastric cancer was significantly associated with tumor size, lymph node metastasis, distance metastasis and TNM stage, and AKR1B10 may be a good prognostic indicator in gastric cancer., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Five phenolic compounds were isolated from the seeds of Perilla (Perilla frutescens L.) using gradient solvent fractionation, silica gel column chromatography, and preparative high-performance liquid chromatography (HPLC). Their chemical structures were identified as caffeic acid-3-O-glucoside (1), rosmarinic acid-3-O-glucoside (2), rosmarinic acid (3), luteolin (4), and apigenin (5) using NMR spectroscopy and HPLC-ESI/MS analysis. Among them, luteolin (4) inhibited α-glucosidase (EC 3.2.1.20) with IC(50) value of 45.4μM. The inhibition kinetic analysed by Dixon plot indicate that luteolin is a noncompetitive inhibitor, and the inhibition constant K(I) was calculated at 45.0μM. Moreover, rosmarinic acid (3) and luteolin (4) inhibited recombinant human aldose reductase (EC 1.1.1.21) with IC(50) values of 11.2 and 0.6μM, respectively. Notably, the inhibition kinetic of luteolin (4) follows a hyperbolic dependence on aldose reductase inhibition by Dixon plot. Thus, inhibition kinetic indicates that luteolin (4) is a mixed-type inhibitor., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)(1) 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing K(m) of 1.3-9.1 μM and k(cat) of 1.1-7.6 min(-1). The stereospecific reduction was also observed in the metabolism of 5α- and 5β-dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background/aims: The detoxification enzyme AKR1B10, a member of the aldo-keto reductase superfamily, is discussed as a new biomarker candidate for hepatocellular carcinoma (HCC). Only rare clinicopathological data on AKRB1B10 in HCC exist. This retrospective study determines the diagnostic and prognostic relevance of AKR1B10 expression in HCC and its relationship to a series of clinicopathological parameters including underlying aetiological factors., Methods: A series of 168 patients with HCCs treated either by surgical resection (n=92) or liver transplantation (n=76) were investigated after construction of a tissue micro-array. Immunohistochemically confirmed AKR1B10 expression was correlated with clinicopathologically relevant parameters as well as proliferative activity (indicated by Ki-67 immunostaining) and apoptosis (terminal deoxyribonucleotide transferase-mediated dUTP nick-end labelling)., Results: AKR1B10 overexpression is significantly associated with lower pT-classification (P=0.030) and highly statistically associated with an underlying viral hepatitis (P<0.001) and the presence of cirrhosis (P<0.001). In addition, loss of AKR1B10 expression correlates with increased proliferative activity (Ki-67, P=0.001). Kaplan-Meier survival analysis of the resection group reveals a poorer prognosis in patients with AKR1B10-negative HCCs compared with patients with strongly positive HCCs (P=0.046)., Conclusions: This study confirms and expands data on the expression of AKR1B10 in HCC, suggesting that this enzyme is a valuable novel biomarker candidate for staging of HCC, especially in patients with underlying virus hepatitis or cirrhosis, and may present a new therapeutic target for multimodal therapy concepts. We confirm its prognostic value and conclude that high expression of AKR1B10 reflects a less aggressive tumour behaviour., (© 2011 John Wiley & Sons A/S.)

Objective: To explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism., Methods: The thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h: control group (C, 5.5 mmol/L), constant high glucose group (HG, 30 mmol/L) and glucose fluctuation (GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitol dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR., Results: The membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group (all P < 0.01) and significantly lower in GF group than those in HG group (all P < 0.01). Sorbierite, AR and AGEs concentrations were significantly higher in HG and GF groups than those in control group (all P < 0.01) and AR and AGEs concentrations were significantly higher in GF group than that in HG group (all P < 0.01). SDH of endothelial cells in HG or GF group were decreased compared with the control group and lower in GF group than in HG group (all P < 0.05). In addition, the mRNA levels of RAGE, eNOS and ET-1 were significantly upregulated compared with the control group (all P < 0.01)., Conclusions: Glucose concentration fluctuation can result in more severe bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1. Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.

Coriander leaves (Coriandrum sativum L.) have become popular worldwide because of their pleasant and delicate aroma. By a hot water extraction method, in which coriander leaves were cut before suspending in boiling water for 2 min, the contents of the main volatile compounds such as alkanals and 2-alkenals from C10 to C14 decreased, while the levels of corresponding alcohols increased in comparison to those obtained by solvent extraction. To investigate the reasons for this variation, an enzyme activity was assayed. By using aliphatic aldehyde as a substrate and NADPH as a coenzyme, strong activity of an aliphatic aldehyde reductase was found for the first time in this herb in the relatively wide pH range of 5.0-9.0, with the maximum activity at pH 8.5. Additionally, the aliphatic aldehyde dehydrogenase, responsible for acid formation, was also found to have a relatively weak activity compared to that of reductase.

AKR1B10 has been identified as a potential biomarker for human nonsmall cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (-)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols.

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41A. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitor's selectivity advantage for AKR1C1 over ALR1.

The incidence of lung cancer (LC) is markedly increased among patients with usual interstitial pneumonia (UIP), and tobacco smoking is its superimposed risk factor. AKR1B10 (aldo-keto reductase 1B10) is frequently overexpressed in pulmonary squamous cell carcinoma and adenocarcinoma in smokers. To investigate the role of AKR1B10 in the pulmonary carcinogenesis in UIP with correlation to tobacco smoking, we examined 13 UIP cases with LC, 13 UIP cases without LC, and 30 cases of non-UIP LC using AKR1B10 immunohistochemistry. AKR1B10 immunoreactivity was confined to squamous metaplasia in honeycomb lesions of UIP and neoplastic cells of LC. Squamous metaplastic foci showed AKR1B10 immunoreactivity more frequently in UIP with LC (24/36 foci, 67%) than in UIP without LC (16/44 foci, 37%) (P<0.01). AKR1B10 expression in UIP was also more frequent in squamous metaplastic foci in smokers (38/67 foci, 57%) than in non-smokers (2/13 foci, 15%) (P<0.01). AKR1B10 expression was frequently observed in both UIP-associated LC (10/13 foci, 77%) and non-UIP LC (18/30 foci, 60%). Ki-67 labeling index was significantly higher in AKR1B10-positive squamous metaplasia of UIP than in AKR1B10-negative squamous metaplasia of UIP. Our results demonstrate that AKR1B10 is involved in the development of LC in UIP in association with smoking. AKR1B10 might be useful as a new marker for identification of high LC risk patients in UIP.

Nine anthraquinones, aurantio-obtusin (1), chryso-obtusin (2), obtusin (3), chryso-obtusin-2-O-beta-D-glucoside (4), physcion (5), emodin (6), chrysophanol (7), obtusifolin (8), and obtusifolin-2-O-beta-D-glucoside (9), isolated from an EtOAc-soluble extract of the seeds of Cassia tora, were subjected to in vitro bioassays to evaluate their inhibitory activity against advanced glycation end products (AGEs) formation and rat lens aldose reductase (RLAR). Among the isolates, compounds 6 and 8 exhibited a significant inhibitory activity on AGEs formation with observed IC(50) values of 118 and 28.9 microM, respectively, in an AGEs-bovine serum albumin (BSA) assay by specific fluorescence. Furthermore, compounds 6 and 8 inhibited AGEs-BSA formation more effectively than aminoguanidine, an AGEs inhibitor, by indirect AGEs-ELISA. N(epsilon)-Carboxymethyllysine (CML)-BSA formation was also inhibited by compounds 6 and 8. Whereas compounds 1, 4, and 6 showed a significant inhibitory activity on RLAR with IC(50) values of 13.6, 8.8, and 15.9 microM, respectively.

AKR1B7 (aldo-keto reductase family 1, member 7; also known as mouse vas deferens protein) is a member of the AKR superfamily, and has been suggested to play a role in detoxifying processes on account of its preferred substrates, 4-hydroxynonenal and isocaproaldehyde. High levels of protein expression were found in the vas deferens and the adrenal gland, where sustained expression is dependent on androgen or ACTH respectively. Recently, a remarkable induction of AKR1B7 expression has been reported in the ovary following exogenous injections of LH. In the present study, we confirm this regulation physiologically during the estrous cycle, observing Akr1b7 expression to be restricted to the theca and stromal cells of the proestrus ovary. To further investigate the role of this detoxifying enzyme in both male and female reproduction, we generated knockout mice deficient in AKR1B7. Although AKR1B7 expression in the vas deferens is considerable and tightly regulated in the ovary of wild-type animals, homozygous mutant animals were found to be viable and no reproductive phenotype was observed. Ovarian follicle maturation and spermatozoa parameters remained normal in the absence of this protein. The determination of serum progesterone revealed an increase in hormone concentration in metestrus, while progesterone was found to be decreased in the estrus phase of the cycle in knockout females.

According to scientific dogma, Saccharomyces cerevisiae cannot grow utilizing xylose as a sole carbon source. Although recombinant DNA technology has overcome this deficiency to some degree, efficient utilization of xylose appears to require complex global changes in gene expression. This complexity provides a significant challenge to the development of yeasts suitable for the utilization of xylose-rich lignocellulosic substrates. In contrast to the dogma, we have found that native strains of S. cerevisiae can grow on xylose as a sole carbon source, albeit very slowly. This observation provided the basis for a new approach using natural selection to develop strains of S. cerevisiae with improved ability to utilize xylose. By applying natural selection and breeding over an extended period, we have developed S. cerevisiae strains that can double in less than 6 h using xylose as a sole carbon source. Strains with improved growth rate possessed increased xylose reductase and xylitol dehydrogenase activities, with the latter showing the greater improvement. This unique, completely nonrecombinant approach to developing xylose-utilizing strains of S. cerevisiae opens an alternative route to the development of yeast that can fully utilize lignocellulosic substrates.

To investigate the underlying causes for aldose reductase deficiency-induced diabetes insipidus, we carried out studies with three genotypic groups of mice. These included wild-type mice, knockout mice, and a newly created bitransgenic line that was homozygous for both the aldose reductase null mutation and an aldose reductase knockin transgene driven by the kidney-specific cadherin promoter to direct transgene expression in the collecting tubule epithelial cells. We found that from early renal developmental stages onward, urine osmolality did not exceed 1,000 mosmol/kgH2O in aldose reductase-deficient mice. The functional defects were correlated with significant renal cellular and structural abnormalities that included cell shrinkage, apoptosis, disorganized tubular and vascular structures, and segmental atrophy. In contrast, the transgenic aldose reductase expression in the bitransgenic mice largely but incompletely rescued urine concentrating capacity and significantly improved renal cell survival, cellular morphology, and renal structures. Together, these results suggest that aldose reductase not only plays important roles in osmoregulation and medullary cell survival but may also be essential for the full maturation of the urine concentrating mechanism.

Activities of aldehyde dehydrogenase and aldehyde reductase in the liver 1.5-, 12-, and 24-month-old rats were measured after 30-min immobilization. Changes in activities of aldehyde dehydrogenase and aldehyde reductase in the liver after immobilization stress depended on animal age. These shifts lead to a strain in endogenous aldehyde utilization in the aldehyde reductase reaction in 1.5-month-old animals and inhibition of utilization of these metabolites in oxidation-reduction reactions in 24-month-old rats.

Renal cell carcinoma (RCC) is one of the most malignant tumors in urology, and due to its insidious onset patients frequently have advanced disease at the time of clinical presentation. Thus, early detection is crucial in management of RCC. To identify tumor specific proteins of RCC, we employed proteomic analysis. We prepared proteins from conventional RCC and the corresponding normal kidney tissues from seven patients with conventional RCC. The expression of proteins was determined by silver stain after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The overall protein expression patterns in the RCC and the normal kidney tissues were quite similar except some areas. Of 66 differentially expressed protein spots (p<0.05 by Student t-test), 8 different proteins from 11 spots were identified by MALDI-TOF-MS. The expression of the following proteins was repressed (p<0.05); aminoacylase-1, enoyl-CoA hydratase, aldehyde reductase, tropomyosin alpha-4 chain, agmatinase and ketohexokinase. Two proteins, vimentin and alpha-1 antitrypsin precursor, were dominantly expressed in RCC (p<0.05).

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mgprot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mgprot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L.h]) after 48 h of fermentation.

An accelerated polyol pathway in diabetes contributes to the development of diabetic complications. To elucidate diabetic nephropathy involving also renal tubular damage, we measured urinary sorbitol concentration concomitantly with urinary N-acetyl-D-glucosaminidase (NAG) excretion in WBN-kob diabetic rats.Twenty-four-hour urinary sorbitol concentrations increased in the diabetic rats in parallel with whole blood sorbitol concentrations. An increase in 24-h urinary NAG excretion coincided with the elevated urinary sorbitol levels in the diabetic rats. The administration of epalrestat, an aldose reductase inhibitor, reduced the increased whole blood and urinary sorbitol concentrations and urinary NAG excretion concomitantly with renal aldose reductase inhibition in the diabetic rats. These results indicate that diabetic nephropathy involves distorted cell function of renal tubules, and that treatment with epalrestat may prevent at least the progress of the nephropathy.

Aim: To examine the relation between aldose reductase (AR) and the development and progression of diabetic retinopathy by comparing the erythrocyte AR levels with the prevalence of diabetic retinopathy in NIDDM patients., Methods: A clinic based cross sectional study was used. 611 NIDDM patients and 73 controls were enrolled. Erythrocyte AR levels were determined by ELISA. These AR levels were then correlated with patient age, duration of diabetes, and HbA(1c) levels. AR levels were also correlated with the prevalence of diabetic retinopathy in the entire NIDDM patient group and in three subgroups formed by separating the NIDDM patients by their duration of diabetes. The prevalence of diabetic retinopathy significantly increased with increased erythrocyte AR levels in patients with duration of diabetes of less than 10 years. A similar, but non-significant correlation between the prevalence of retinopathy and increased erythrocyte AR levels was observed in patients with diabetes duration of 10-20 and >/=20 years., Results: The prevalence of diabetic retinopathy increased with increased erythrocyte AR levels in NIDDM patients with a duration of diabetes of less than 10 years., Conclusion: It was suggested that the inhibition of AR in patients with early NIDDM might be beneficial in reducing the development of diabetic retinopathy.

Newborn rats are not capable of producing concentrated urine. With development of the concentrating system and a hypertonic medullary interstitium, intracellular osmolytes, such as sorbitol, accumulate in the renal medulla. Sorbitol is produced from glucose in a reaction catalyzed by aldose reductase (AR). The purpose of this study was to establish the time of expression and distribution of AR in the developing rat kidney. Kidneys from 16-, 18-, and 20-day-old fetuses and 1-, 3-, 4-, 5-, 7-, 14-, and 21-day-old pups were processed for immunohistochemistry and immunoblot analysis. In adult animals, AR was expressed only in the inner medulla, in which it was localized in ascending thin limbs (ATLs), inner medullary collecting ducts (IMCDs), and interstitial cells. AR immunoreactivity was not detected in fetal kidneys but was observed in the terminal part of the descending thin limb and IMCD in the renal papilla of 1-day-old pups. At birth, all of the loops of Henle are configured as short loops and there are no ATLs. After birth, papillary thick ascending limbs are gradually transformed into ATLs by a process that involves apoptotic deletion of cells from the thick ascending limb. During this time, AR immunoreactivity appeared in the cells undergoing transformation in the ascending limb, beginning at the papillary tip and ascending to the border between the outer medulla and the inner medulla. However, there was no labeling of apoptotic cells. The expression of AR in both the ATL and the IMCD gradually increased during kidney development. We conclude that AR expression in the inner medulla coincides with the increase in medullary tonicity that is known to occur during the first 3 wk after birth. On the basis of the observation that only AR-negative cells were deleted by apoptosis in the differentiating ATL, we propose that AR may protect ATL cells against apoptosis.

Background: The organic osmolyte sorbitol plays an important role in the osmoregulation of immortalized epithelial cells of the thick ascending limb of Henle's loop (TALH) of rabbit. The intracellular sorbitol content seems to depend strongly on the extracellular osmolarity. To investigate the nature of the osmotic regulation we characterized the aldose reductase., Methods: We determined aldose reductase activity enzymatically and the content of organic osmolytes by HPLC., Results: The aldose reductase activity correlates with the extracellular tonicity. Elevating the osmolarity of the medium from 300 to 600 mosm/l by addition of NaCl or sucrose resulted in a significant increase of maximal velocity (V(max)) of the adapted cells from 8 +/- 1 micromol/g x min (300 mosm/l) to 322 +/- 28 micromol/g x min (600 mosm/l, NaCl) or 54 +/- 9 micromol/g x min (600 mosm/l, sucrose), respectively, while affinity (K(m)) remained unchanged. But we found no rise of aldose reductase activity when extracellular urea concentration was elevated. Similar alterations in V(max) were observed when the activity of the highly enriched enzyme was determined with glucose as substrate. Elevation of the extracellular osmolarity by NaCl and sucrose strongly induced the expression of aldose reductase protein with an apparent molecular weight of 39 kD. The affinity of glucose is characteristically low with a K(m) above 300 mmol/l. Aldose reductase utilizes both NADPH and with lower affinity NADH as coenzymes. In vitro sulfate ions (0.4 mol/l) results in a two-fold activation of the aldose reductase activity whereas sodium (200-400 mmol/l) decreased the activity significantly (22-33%). Potassium and chloride up to 400 mmol/l did not alter the aldose reductase activity in vitro., Conclusions: These results indicate that the aldose reductase of TALH cells of the outer medulla is osmotically regulated and has many similarities with aldose reductase in renal inner medulla. Therefore, intracellular sorbitol synthesis seems to be of similar importance in the osmoregulation of TALH cells as in the inner medulla., (Copyright 2001 S. Karger AG, Basel)

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.

The reversibility of S-thiolation of aldose reductase was shown in intact bovine lens subjected to oxidative stress. The glutathione modified aldose reductase generated in the lens as a consequence of hyperbaric oxygen treatment was recovered in its reduced form following culturing in normobaric air conditions. Nucleus and cortex were differently affected by both oxidative treatment and normobaric air recovery. The extent of S-thiolation of aldose reductase appeared to be higher in the nucleus than in the cortex. Moreover, the nucleus, but not the cortex, was unable to completely recover from the protein S-thiolation process. The ratios of GSH/GSSG and NADPH/NADP(+)as well as the Energy Charge values were determined in the cortex and nucleus both after oxidative stress and recovery. The results are consistent with the existence of a quite well-defined boundary between the two lens regions. Moreover, they are supportive of the hypothesis that thiol/disulfide exchange has the potential to be a regulatory mechanism for certain enzymes which can modulate the flux of NADPH inside the cell., (Copyright 2000 Academic Press.)

Purpose: Sugars and polyols content assessment in cataractous nucleus in diabetics and non-diabetics., Material and Methods: Cataractous nucleus was obtained during extracapsular cataract extraction derived from 52 diabetic and 58 non-diabetic patients. The content of glucose, fructose, sorbitol and mioinositol in examined material was determined using iquid gas chromatography., Results: Average contents of sorbitol, glucose and fructose in cataractous nucleus diabetic patients (ZSC) are significantly higher than in non-diabetics (ZS). No changes were observed in mioinositol content in both studied groups., Conclusion: The role of the polyol pathway in the development of diabetic cataract is considerable.

It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region.

2-Iodohexadecanal (2-IHDA) is a major thyroid iodolipid. It mimics the main regulatory effects of iodide on thyroid metabolism: inhibition of H2O2 production and of adenylyl cyclase. The biosynthesis of 2-IHDA and its metabolism have been investigated in cultured dog thyroid cells maintained in a differentiated state by forskolin. Incubation of these cells with [9,10-3H]hexadecan-1-ol or [9,10-3H]palmitic acid labeled several phospholipids, but [9, 10-3H]hexadecan-1-ol was selectively incorporated into plasmenylethanolamine. In the presence of an exogenous H2O2 generating system (glucose oxidase), iodide induced the production of [9,10-3H]2-IHDA from [9,10-3H]hexadecan-1-ol-labeled cells but not from [9,10-3H]palmitic acid-labeled cells. 2-IHDA was also generated during the lactoperoxidase-catalyzed iodination of brain and heart plasmalogens, and of ethyl hexadec-1-enyl ether, a synthetic vinyl ether-containing compound. Taken together, these results show that thyroid 2-IHDA is derived from plasmenylethanolamine via an attack of reactive iodine on the vinyl ether group. 2-Iodohexadecan-1-ol (2-IHDO) was also detected in these studies; it was formed later than 2-IHDA, and thyroid cells converted exogenous 2-IHDA into 2-IHDO in a time-dependent way. The ratio of 2-IHDO/2-IHDA increased with H2O2 production and decreased as a function of iodide concentration. An aldehyde-reducing activity was detected in subcellular fractions of the horse thyroid. No formation of 2-iodohexadecanoic acid could be detected. Reduction into the biologically inactive 2-IHDO is thus a major metabolic pathway of 2-IHDA in dog thyrocytes.

Hypercalcemia is often associated with a urinary concentration defect. During antidiuresis, organic osmolytes [sorbitol, myo-inositol, taurine, and glycerophosphorylcholine (GPC)] accumulate in the renal inner medulla and are essential for urinary concentration. To clarify the relationship between organic osmolytes and urinary concentration defect in hypercalcemia, examination was made of the effects of hypercalcemia on renal medullary osmolytes content. Rats were put in a state of hypercalcemia by a calcium-rich diet supplemented with CaCO3 (2.5%/wt) and daily s.c. injection of 1.25(OH)2VitD3 (1.6 micrograms/kg). They were killed on days 7 and 14. Hypercalcemia induced a urinary concentration defect. Myo-inositol, sorbitol, and GPC contents in the renal medulla were significantly reduced. Aldose reductase activity decreased significantly. Hypercalcemia would thus appear to directly affect renal medullary content of organic osmolytes, thereby modifying renal concentration ability.

Skeletal muscle contains high levels of aldose reductase that catalyzes the reduction of galactose to the polyol galactitol. Galactitol and water were measured in muscle of rats fed a high galactose diet with or without addition of the aldose reductase inhibitor sorbinil. Galactitol, measured in isolated samples of muscle by HPLC, reached steady-state levels (5.9 +/- 1.0 mg/g tissue) within 3 days. Muscle water, determined in vivo by magnetic resonance imaging, increased (51 +/- 5%, P < 0.02) to steady-state levels within 7 days. Both the increased galactitol and water remained constant for the 4-month duration of this study. Aldose reductase activity also remained constant. Sorbinil prevented both the increase in galactitol and the increase in water. These results suggest that the increase in water is due to the osmotic effects of galactitol accumulation and demonstrate that galactitol and water accumulation neither up-regulate nor down-regulate aldose reductase expression in skeletal muscle.

Tumor-associated protein variants were detected by two-dimensional gel electrophoresis (2-DE) in soluble proteins from chemically induced rat hepatomas and transformed rat liver cell lines. Among them, a series of 8 protein variants was found localized at similar sites in 2-DE gels (33-35 kDa, with different pI values). We characterized four of them. In situ peptide mapping with limited proteolysis disclosed a significant relationship between these four individual protein spots. Their different position in 2-DE gels might be due to posttranslational modifications: one of the variants was phosphorylated, three others were modified by glycosylation. The most prominent tumor-associated protein variant of this series (spot 17) was further studied by amino acid analysis and internal amino acid microsequencing. It became evident that this variant is identical to aldose reductase (EC 1.1.1.21). This enzyme of the sorbitol pathway is expressed in the liver during embryogenesis, but is absent in adult rat liver. Our results suggest that it is reexpressed and functionally active during liver carcinogenesis.

Blood-retinal barrier (BRB) breakdown occurs in human diabetic retinopathy and can lead to significant loss of vision. The galactosemic rat serves as a model for human diabetic retinopathy and develops many of the same ocular complications, including BRB failure. Aldose reductase (AR), an enzyme in the polyol pathway, has been implicated in several of these complications. Electron microscopic immunocytochemical staining for albumin can be used to visualize extravasated albumin in control and galactosemic rats with and without AR inhibition to reveal the mechanism for galactosemia-related BRB failure without the use of tracer substances, and to determine whether AR activity influences this mechanism. Electron microscopic immunocytochemical labeling of AR can reveal the cellular distribution of AR in normal and galactosemic rat retina and determine whether a correlation exists between BRB breakdown and AR expression in cells that form or influence the BRB. Extravascular albumin is demonstrable as early as 2.5 months in galactosemic rats, which is prior to the presentation of ultrastructural changes. Albumin-positivity is visualized in retinal vascular endothelial (RVE) cell cytoplasm from galactosemic rats both diffusely and within vesicles, but it is not detected in comparably aged normal rat RVE cells or within the tight junctions of the RVE or RPE (the cells that form the BRB) in control or galactosemic rats, suggesting that BRB breakdown in galactosemic rats may be mediated through two mechanisms: a permeation of the RVE cell membrane leading to diffuse cytoplasmic positivity for albumin, and vesicular transport across the RVE. The AR inhibitor, sorbinil, prevents diffuse cytoplasmic staining of RVE cells, but not vesicular staining, implying that RVE membrane permeability may be altered by AR activity, but vesicle formation does not appear to be affected. AR was immunocytochemically demonstrated in some RVE cells from galactosemic rats, but not from controls. In perivascular astrocytes, which may influence the integrity of the inner BRB, AR labeling was augmented in galactosemic rats. Increased AR expression in RVE cells and perivascular astrocytes from galactosemic rats provides additional support for a role for AR in galactosemia-related BRB failure.

Sugar alcohols have been reported to accumulate in retinal pigment epithelium (RPE) of diabetic animals. This finding has raised interest in the role of RPE in diabetes-associated retinal changes such as cystoid macular edema. To confirm the presence of aldose reductase in this tissue, the NADPH-dependent enzyme was purified to an apparent homogeneity from cultured human RPE cells, characterized, and its biochemical properties investigated. The induction of aldose reductase by hypertonic stress was also examined. The purification of aldose reductase was performed by a series of chromatographic steps which include gel filtration, affinity chromatography and chromatofocusing. Final purity achieved was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic properties and susceptibility to inhibition of the purified aldose reductase were essentially identical to aldose reductase purified from human placenta and kidney. In addition to aldose reductase, chromatofocusing demonstrated the presence of aldehyde reductase, another NADPH-dependent reductase. However, the amounts of aldehyde reductase present were much smaller than those of aldose reductase and the levels of aldehyde reductase appeared too small to contribute to the polyol production in the RPE cells. Culture of RPE cells in hypertonic medium containing 150 mM sodium chloride (600 mosmol total) increased both reductase activity, monitored with DL-glyceraldehyde as substrate, and immunoblot staining for aldose reductase. Chromatofocusing of RPE cells cultured in hypertonic media resulted in a prominent increase in the peak corresponding to aldose reductase compared to the peak height of cells grown in control medium. No increase in aldehyde reductase from RPE cells cultured in hypertonic medium was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

An antibody-sandwich enzyme-linked immunosorbent assay (ELISA) for evaluating tissue levels of aldose reductase was developed using a polyclonal antibody prepared against the recombinant enzyme expressed in a baculovirus system. The specificity of this antibody to aldose reductase was verified by immunoprecipitation, immunoblotting and ELISA. The polyclonal antibody did not crossreact with human aldehyde reductase, an enzyme in the same aldo-keto reductase family structurally and functionally related to aldose reductase. The sensitivity and specificity of this assay method enabled direct determination of aldose reductase level in various human tissues including the erythrocyte. The highest level of aldose reductase was detected in the kidney medulla among tissues investigated. More than a 2-fold variability in the erythrocyte aldose reductase was demonstrated among healthy individuals, indicating the heterogeneity of this enzyme expression in a human population. This assay system may be useful for direct measurement of the level of tissue aldose reductase in conjunction with the evaluation of the efficacy of aldose reductase inhibitors prescribed for the treatment of diabetic complications.

4-Hydroxynonenal (4-HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4-HNE are related to the quantity formed in the cells and to the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4-HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation. In hepatoma cells, generally, 4-HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4-HNE metabolism was mainly due to alcohol dehydrogenase and glutathione-S-transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH-130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally-added 4-HNE in hepatoma cells was proportional to their content of 4-HNE metabolizing enzymes.

There is increasing evidence that a link between the polyol pathway and prostaglandins is important in the pathogenesis of diabetic nephropathy. The presence of the polyol pathway in the kidneys of normal animals, the galactose-fed rat, and animals with experimental diabetes has been established. While aldose reductase (AR) immunoreactive protein (AR-IRP) and AR mRNA are expressed at high levels in renal medulla, the sites of AR synthesis and regulation and metabolic consequences of AR activity in renal cortex are uncertain. The present study was conducted to test the hypothesis that AR expression and PGE2 production are coordinately regulated in glomerular mesangial cells. To test this hypothesis, we measured AR-IRP, AR mRNA, and PGE2 production in mesangial cells isolated from rats maintained on diets containing normal chow (MC-N), 50% galactose (MC-G), and 50% dextrin (MC-D). The rank order for each parameter studied (AR-IRP, AR mRNA, PGE2) was MC-N > MC-G > MC-D. Western blot analysis demonstrated that MC-N (optical density [OD] 1.0), MC-G (OD 0.59), and MC-D (OD 0.25) express AR-IRP. Slot-blot analyses demonstrated that levels of AR mRNA were greatest in MC-N (1.0), intermediate in MC-G (0.49), and lowest in MC-D (0.31). Ribonuclease (RNase) protection analyses demonstrated a similar pattern of AR mRNA expression, with MC-N at 1.0, MC-G at 0.60, and MC-D at 0.33. PGE2 production (pg/5 x 10(4) cells/30 min) was highest in MC-N (278 +/- 29), intermediate in MC-G (110 +/- 9), and lowest in MC-D (37 +/- 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.

Pharmacological effects of Gosha-jinki-gan-ryo extract (KJE) on experimental diabetes induced by cyproheptadine (CPH), aldose reductase activity, and experimental peripheral neuropathy were studied. The effects of KJE were compared with those of Hachimi-jio-gan-ryo extract (HJE). KJE at 417 mg/kg/day (5 times the daily dose in humans) and HJE at 367 mg/kg/day (5 times the daily dose in humans) significantly inhibited the decrease in glucose tolerance by CPH. KJE and HJE inhibited aldose reductase activity, when DL-glyceraldehyde was used as substrate, with IC50 values of 2.68 x 10(-5) g/ml and 4.45 x 10(-5) g/ml, respectively and when D-glucose was used as substrate, with IC50 values of 1.04 x 10(-4) g/ml and 1.55 x 10(-4) g/ml, respectively. KJE at 209 mg/kg/day (2.5 times the daily dose in humans) and HJE at 367 mg/kg/day significantly reduced peripheral neuropathy induced by crushing the sciatic nerve in rats. The potency of these effects of KJE was stronger than that of HJE, when a comparison was made on the basis of the daily dose.