Microtubular protein impairment by pentanal and hexanal

Pentanal and hexanal are some of the aldehydes produced by lipid peroxidation, that causes damage to several subcellular structures. Lipoperoxidation products may directly attack cytoskeleton structures, the integrity of which is required for secretion mechanisms, e.g. 4-hydroxy-alkenals after microtubular integrity and function. Purified microtubular protein incubated with pentanal and hexanal at different concentrations revealed a tubulin-aldehyde interaction affecting the polymerization reaction and the colchicine-binding activity. These reactions apparently do not involve sulphydryl groups, and the addition of mercaptoethanol does not protect microtubules from the action of aldehydes, the effect of which is however more homogeneous, as only small differences can be noticed among the various aldehyde concentrations used.     

The relationship of calcium and parathyroid hormone in their effect on heart cells

Parathyroid hormone (PTH), the plasma concentration of which is raised in uremia, has been suggested as one of the agents responsible for the myocardial changes commonly seen in uremia. The effect of intact [1–84] PTH on rat heart cells grown in tissue culture has been studied. Addition of the hormone to the media significantly stimulated beating rate. The stimulation was directly proportional to the amount of PTH in the medium. Excessively high concentration of PTH caused immediate cessation of the beating, which was reversed by the addition of calcium to the medium. The extent of stimulation by PTH was inversely proportional to the calcium concentrations in the medium. Isoproterenol and phenylephrine at excessively high concentrations in the medium did not mimic the PTH effect either alone or together with PTH. When beating ceased due to verapamil the effect was not reversed by the addition of calcium to the medium.Calcium added to the myocytes seen after beating ceased reversed the effect and the cells started to beat again. Cells kept for a longer period in the arrested state were not revived by the addition of calcium.     

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days (‘indefinite’ survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.     

Spectroscopic studies of methylglyoxal in water and dimethylsulfoxide

Methylglyoxal is a highly reactive dicarbonyl compound, which reacts in vivo with biological macromolecules and thereby affects their structure and function. These changes are associated with complications during aging, diabetes and Alzheimer's disease as well as with growth inhibition in different tumors. Many enzymes are involved in the metabolism of methylglyoxal, but its true physiological role in metabolism and chemical properties are still obscure. In this study it was shown that methylglyoxal, during the freeze-drying of aqueous solutions, polymerizes into small polymeric structures which are stable in organic media such as dimethylsulfoxide. When re-exposed to water, the polymers are immediately transformed into the monomeric mono- and dihydrate forms of methylglyoxal. By NMR and UV spectroscopy, it was shown that solvent, temperature, and the amount of available water strongly influence the equilibrium of the different forms of methylglyoxal and thereby change its reactivity. 1H and 13C NMR spectroscopy were used to determine the structures of the different monomeric and oligomeric structures of methylglyoxal.     

Aldehyde-induced modifications of the microtubular system in 3T3 fibroblasts.

The molecular structure of aldehydes is closely related to their antimicrotubular effect. Morphological modifications of the microtubular system in living cells after incubation with certain aldehydes are consistent with biochemical alterations detected in previous research. The microtubular arrangement was visualized by an immunofluorescence technique with antitubulin antibodies, while the content of tubulin in the cells was evaluated by a colchicine binding assay. 2-Nonenal behaved similarly to 4-hydroxynonenal, a lipid peroxidation product, disorganizing microtubular network in 3T3 fibroblasts and decreasing the amounts of tubulin able to bind labelled colchicine. Nonanal did not significantly impair the tubulin characteristics in the cells, despite the fact that it has been shown to be active on the purified microtubular system; benzaldehyde was ineffective. This would appear to explain the mechanisms of interaction of aliphatic aldehydes which might be suitable for use as antimicrotubular drugs.     

The possible role of methylglyoxal metabolism in cancer

Tumours reprogram their metabolism to acquire an evolutionary advantage over normal cells. However, not all such metabolic pathways support energy production. An example of these metabolic pathways is the Methylglyoxal (MG) one. This pathway helps maintain the redox state, and it might act as a phosphate sensor that monitors the intracellular phosphate levels. In this work, we discuss the biochemical step of the MG pathway and interrelate it with cancer.     

Discovery of new quinolines as potent colchicine binding site inhibitors: design,synthesis, docking studies,and anti-proliferative evaluation

Discovering of new anticancer agents with potential activity against tubulin polymerisation is still a promising approach. Colchicine binding site inhibitors are the most relevant anti-tubulin polymerisation agents. Thus, new quinoline derivatives have been designed and synthesised to possess the same essential pharmacophoric features of colchicine binding site inhibitors. The synthesised compounds were tested in vitro against a panel of three human cancer cell lines (HepG-2, HCT-116, and MCF-7) using colchicine as a positive control. Comparing to colchicine (IC₅₀ = 7.40, 9.32, and 10.41 µM against HepG-2, HCT-116, and MCF-7, respectively), compounds 20, 21, 22, 23, 24, 25, 26, and 28 exhibited superior cytotoxic activities with IC₅₀ values ranging from 1.78 to 9.19 µM. In order to sightsee the proposed mechanism of anti-proliferative activity, the most active members were further evaluated in vitro for their inhibitory activities against tubulin polymerisation. Compounds 21 and 32 exhibited the highest tubulin polymerisation inhibitory effect with IC₅₀ values of 9.11 and 10.5 nM, respectively. Such members showed activities higher than that of colchicine (IC₅₀ = 10.6 nM) and CA-4 (IC₅₀ = 13.2 nM). The impact of the most promising compound 25 on cell cycle distribution was assessed. The results revealed that compound 25 can arrest the cell cycle at G2/M phase. Annexin V and PI double staining assay was carried out to explore the apoptotic effect of the synthesised compounds. Compound 25 induced apoptotic effect on HepG-2 thirteen times more than the control cells. To examine the binding pattern of the target compounds against the tubulin heterodimers active site, molecular docking studies were carried out.     

Characteristic effects of methylglyoxal and its degraded product formate on viability of human histiocytes: a possible detoxification pathway of methylglyoxal

Methylglyoxal (MGO) is a toxic and highly reactive alpha-oxoaldehyde, elevated in the states of various diseases underlying enhanced oxidative stress. Furthermore, MGO has been reported to generate another aldehyde, formic acid (FA). In this sense, investigating the biological property of FA is crucially important. The present study examined the effects of MGO and FA on cell viability using the U937 human histiocytic cell line. FA showed a dose-dependent increase in cell viability at the concentrations of MGO in which cell viability decreased. The mechanism of the increase by FA involved the presence of endogenous hydrogen peroxide (H₂O₂) and tetrahydrofolate in the folate pathway, whereas that of the decrease in cell viability by MGO involved interaction with H₂O₂ and oxidative damage. These findings suggest that FA production by MGO degradation may play a role in attenuation of oxidative cellular injury caused by MGO. We hypothesize that FA generation pathway constitutes a detoxification system for MGO.     

Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC₅₀) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (≥2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [¹⁴C]-labelled species) showed that adduct formation was much greater for iso-VPA-G. When [¹⁴C]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides.     

基于微管蛋白和秋水仙碱结合位点化合物构建3D-药效团新模型及先导物虚拟筛选

基于作用于微管蛋白秋水仙碱结合位点的小分子抑制剂与生物靶标的复合晶体结构,采用分子模拟软件Discovery Studio 3.0的受体-配体药效团产生程序建立了系列3D药效团新模型（M1-M6）,并用20个已知微管抑制剂验证了其可靠性.用新药效团模型对约10000个化合物的数据库进行了虚拟筛选,发现了一些潜在先导物.据此合成的二芳烃胺类新化合物20在抑制人白血细胞K562的初步实验中显示出了明显的细胞形态变化和抑制活性,对多种人癌细胞A549,KB,KBvin和DU145均有较强的抑制活性（GI500.17～1.02μmol/L）,表明以此新构建的药效团模型进行理性设计和寻找新型抗癌先导物的方法具有一定的可行性.     

Isoflurane’s effect on interfacial dynamics in GAPDH influences methylglyoxal reactivity

Diabetic surgical patients are at risk for peri- and post-operative complications, which can be prevented by maintaining tight glycemic control during anesthesia. Control of blood sugar would decrease unwanted chemical reactions, such as protein glycation, minimizing tissue dysfunction. Methylglyoxal (MG) is a major contributor to protein modification and tissue dysfunction seen in diabetic patients. We hypothesized that inhaled anesthetics may play a role in protein glycation and examined the effects of isoflurane on MG-induced modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Isoflurane promoted MG-induced modification of GAPDH as evidenced by an increase in fluorescent glycation products, a change in chromatographic elution patterns and a loss of enzyme activity. Isoflurane’s effect may be mediated by altering interfacial events. Our working model involves the binding of isoflurane to GAPDH, increasing the susceptibility to MG-induced modification of residues involved in oligomerization. These findings suggest a molecular basis for maintaining glycemic control during anesthesia.     

The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey

Methylglyoxal in New Zealand manuka honey has been shown to originate from dihydroxyacetone, which is present in the nectar of manuka flowers in varying amounts. Manuka honey, which was freshly produced by bees, contained low levels of methylglyoxal and high levels of dihydroxyacetone. Storage of these honeys at 37 °C led to a decrease in the dihydroxyacetone content and a related increase in methylglyoxal. Addition of dihydroxyacetone to clover honey followed by incubation resulted in methylglyoxal levels similar to those found in manuka honey. Nectar washed from manuka flowers contained high levels of dihydroxyacetone and no detectable methylglyoxal.     

Cell cycle regulation of the microtubular cytoskeleton

The microtubular element of the plant cytoskeleton undergoes dramatic architectural changes in the course of the cell cycle, specifically at the entry into and exit from mitosis. These changes underlie the acquisition of specialized properties and functions involved, for example, in the equal segregation of chromosomes and the correct positioning and formation of the new cell wall. Here we review some of the molecular mechanisms by which the dynamics and the organization of microtubules are regulated and suggest how these mechanisms may be under the control of cell cycle events.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Characterization of gangliosides from Ehrlich ascites tumour cells and their variants

Differences in the nature of the gangliosides present in two types of Ehrlich ascites tumour (EAT) cells, the adherent and non-adherent EAT cells, were studied. Gangliosides were isolated by DEAE Sephadex column chromatography and analysed by high-performance thin-layer chromatography (HPTLC). The non-adherent EAT (na-EAT) cells which grow in the peritoneal cavity of mice were selected for growth on basement membrane and tissue culture plastic to give the adherent EAT (a-EAT) cells. na-EAT cells contained 1.57 nmol lipid-bound sialic acid per mg protein and at least 12 different gangliosides, including major gangliosides such as GM3, GM2, GM1, GD3, GD1a and GT1b. On the other hand, the ganglioside pattern of a-EAT cells differed significantly from that of na-EAT cells, both quantitatively and qualitatively. The content of lipid-bound sialic acid in a-EAT cells was only 0.24 nmol per mg of protein. The gangliosides in a-EAT cells were characterized as GD1a and trisialogangliosides and, significantly, a-EAT cells did not contain monosialogangliosides. Neutral glycolipids were isolated from both cell lines and their patterns were compared. In contrast to the gangliosides pattern, their neutral glycolipid patterns were similar. Glucosylceramide and lactosylceramide were the major components in both types of cells. In addition to na- and a-EAT cells, a-EAT cells were passaged in mice by intraperitoneal injection, giving rise to a third variant (c/m EAT cells). We analysed the gangliosides in c/m EAT cells to determine whether there was a change in the ganglioside pattern found in na-EAT cells. After repeated passage of c/m EAT cells in mice, the pattern of gangliosides shifted to that of na-EAT cells. Alterations of ganglioside composition may be associated with the growth environment of the murine peritoneal cavity; alternatively, a selection process may have occurred.Abbreviations EAT cells Ehrlich ascites tumour cells - na-EAT cells non-adherent EAT cells - a-EAT cells adherent EAT cells - c/m EAT cells cultured a-EAT cells passaged in mice - HPTLC high-performance thin-layer chromatography - PBS 10 mM phosphate-buffered saline, pH 7.2, containing 0.15 M NaCl - EDTA ethylene-diaminetetraacetic acid - TFA trifluoroacetic acid - TG thioglycollate - Cer ceramide (N-fatty acyl sphingosine) - GM3 NeuAc2-3Gal1-4Glc-Cer - GM2 GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GM1a Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GD3 NeuAc2-8NeuAc2-3Gal1-4Glc-Cer - GD1a NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-Cer - GT1b NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Glc-Cer - LacCer Gal1-4Glc-Cer - Gb3 Gal1-4Gal1-4Glc-Cer - Gb4 GalNAc1-4Gal1-4Gal1-4Glc-Cer This paper is dedicated to my esteemed colleague, Sen-itiroh Hakomori on the occasion of his 65th birthday.     

The action of cytochalasin A on the in vitro polymerization of brain tubulin and muscle G-actin

The presence of cytochalasin A inhibits the self-assembly of beef brain tubulin and rabbit muscle G-actin in vitro and also decreases the colchicine binding of tubulin. Prior reaction of cytochalasin A with 2-mercaptoethanol destroys its inhibitory effects. It is shown that cytochalasin A exerts its actions by reacting with sulfhydryl groups, possibly causing irreversible structural changes in the proteins. Cytochalasin B does not affect the tubulin assembly reaction.     

Oxidative damage of DNA induced by the reaction of methylglyoxal with lysine in the presence of ferritin

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin. [BMB Reports 2013; 46(4): 225-229]     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

Accumulation of endogenous methylglyoxal impaired insulin signaling in adipose tissue of fructose-fed rats

Increased accumulation of methylglyoxal (MG) has been linked to different insulin resistance states including diabetes and hypertension. In this study, the effects of MG on insulin signaling pathway were investigated. Following 9 weeks of fructose treatment, an insulin resistance state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, high blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. More importantly, we observed a close correlation between the development of insulin resistance and elevated MG level in serum and adipose tissue. Both insulin resistance state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC). When 3T3-L1 adipocytes were treated directly with MG, the impaired insulin signaling was also observed, indicated by decreased insulin-induced insulin-receptor substrate-1 (IRS-1) tyrosine phosphorylation and the decreased kinase activity of phosphatidylinositol (PI) 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In conclusion, the increase in endogenous MG accumulation impairs insulin-signaling pathway and decreases insulin-stimulated glucose uptake in adipose tissue, which may contribute to the development of insulin resistance.     

Effects of sodium fluoride (NaF) on the cilia and microtubular system of Tetrahymena

The effect of the nucleophilic reagent NaF on the microtubular system of Tetrahymena was studied by using scanning electron microscopy (SEM), confocal microscopy, and flow cytometry. Treatments with 40 mM NaF significantly reduced the amount of alpha-tubulin while 80 mM treatment did not alter its quantity. One possible explanation for this alpha-tubulin overexpression is that the higher amount of alpha-tubulin enables this organism to carry out the appropriate function of the cytoskeleton under this undesirable influence of higher amounts of 80 nM NaF. However, the amount of acetylated tubulin increased in a dose-dependent manner. The cilia became fragile under the effect of 80 mM NaF. Confocal microscopy revealed that after 40 mM NaF treatment transversal microtubule bands (TMs) and longitudinal microtubule bands (LMs) as well as basal bodies (BBs) were extremely strong decorated with anti-acetylated tubulin antibody and TM-localization abnormalities were visible. In the 80 mM NaF-treated cells, the deep fiber of oral apparatus was very strongly labeled, while the TMs and LMs were less decorated with anti-acetylated tubulin antibody, and LM deformities were visible. It is supposed that post-translational tubulin modifications (e.g., acetylation) defend the microtubules against the NaF-induced injury. NaF is able to influence the activity of several enzymes and G-proteins, therefore is capable to alter the structure, metabolism, and the dynamics of microtubular system. The possible connection of signaling and cytoskeletal system in Tetrahymena is discussed.