31P and 3-fluoro-3-deoxy-D-glucose 19F in vivo NMR spectroscopy of aged rat brain

31P and 3-fluoro-3-deoxy-D-glucose (3-FDG) 19F nuclear magnetic resonance (NMR) spectroscopic investigations were performed on aged rat brain in vivo. Phosphodiester (PDE) was found to be significantly higher (p less than 0.05) in aged brain (18 months, n = 10) than adult controls (6 months, n = 10) suggesting accelerated phospholipid catabolism in aged brain. Inorganic phosphate (Pi) levels were significantly lower (p less than 0.05) in aged brain (n = 5) than in normal adult controls (n = 5). The sorbitol index, a measure of aldose reductase activities in vivo, determined by the 3-FDG 19F NMR method, revealed that aldose reductase activities were significantly higher (p less than 0.05) in aged brain.     

Inhibition by nonsteroidal antiinflammatory drugs of luminol-dependent human-granulocyte chemiluminescence and [3H]FMLP binding

A system is described to evaluate for nonsteroidal antiinflammatory drugs by means of luminol-dependent human-granulocyte chemiluminescence (CL) is described. The CL is produced using either opsonized zymosan (yeast cells) or the soluble chemotactic peptide f-Met-Leu-Phe as the perturbant of the granulocyte membrane. Using either system, the following drug effects 2×10^–5 M were noted: only sulindac sulfide, and not sulindac sulfone or sulindac, displayed marked inhibition of chemiluminescence, following the in vivo data regarding inflammatory effects. The 5-OH indomethacin metabolite was likewise inactive as an inhibitor of CL mirroring in vivo effects. MK(+)410, MK(–)830 and MK835 all showed approximately 50% inhibition of CL, displaying deviation from in vivo data. MK(+)830 markedly stimulated CL, 4–6 times the control (without drug), which is clearly different from its enantiomer, MK(–)830. The reasons for this behavior are unclear. However, receptor binding studies with [³H]FMLP were accomplished in the presence and absence of the various drugs at 2×10⁵ M that were effective inhibitors of chemiluminescence (CL). Indomethacin, MK(–)830 and MK(+)410 had equivalent percent control binding and percent control CL. Sulindac sulfide and MK(+)835 both had higher percent control binding than percent control CL, with MK(+)835 displaying apparent increased numbers of available receptors relative to control. MK(+)830, which produces large increases in CL, produced a minor effect on percent control binding. A direct relationship between binding and CL does not exist with each drug. Chemiluminescence is dependent on ion movement and oxidative metabolism and is a secondary event to agonist-receptor occupation.     

The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions

The enzyme aldose reductase plays an important role in the osmo-protection mechanism of diverse organisms. Here, we show that yeast aldose reductase is encoded by the GRE3 gene. Expression of GRE3 is carbon-source independent and up-regulated by different stress conditions, such as NaCl, H2O2, 39 degrees C and carbon starvation. Measurements of enzyme activity and intracellular sorbitol in wild-type cells also indicate that yeast aldose reductase is stress-regulated. Overexpression of GRE3 increases methylglyoxal tolerance in Saccharomyces cerevisiae. Furthermore, high expression of GRE3 complements the deficiency of the glyoxalase system of a glo1delta mutant strain. Consistent with this, in vitro and in vivo assays of yeast aldose reductase activity indicate that methylglyoxal is an endogenous substrate of aldose reductase. Furthermore, addition of NaCl or H2O2 to exponential-phase cells triggers an initial transient increase in the intracellular level of methylglyoxal, which is dependent on the Gre3p and Glo1p function. These observations indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methylglyoxal degradative pathway, in which this compound is metabolised by the action of aldose reductase.     

Regulation of LFA-3 (CD58) by dexamethasone and retinoic acids in vitro

INTRODUCTION: The oral form of lichen planus (OLP) is a chronic inflammatory disease characterized by the accumulation of T cells below the basal layer of the buccal mucosa. We have previously shown that the adhesion molecule lymphocyte function-associated antigen-3 (LFA-3; CD58) is up-regulated in lesional tissue and may play an important part in the molecular pathology of this disease. OLP is often treated with glucocorticoids, and glucocorticoid-resistant cases with retinoid drugs. However, it is not yet known whether the pharmacological action of these drugs is mediated directly or indirectly via LFA-3. METHODS: In the present experiments we have used the technique of flow cytometry (FCM) to accurately measure the effects of the glucocorticoid dexamethasone (DEX), all-trans retinoic acid (ATRA) and 13-cis retinoic acid (13RA) on LFA-3 expression by three main cell types found in OLP lesions--an oral epithelial cell line (KB cells), a T lymphocyte cell line (Jurkat cells) and an antigen presenting myelomonocytic cell line (U937 cells). The relative levels of the specific receptors for the drugs were also determined in these cell lines using FCM. RESULTS: ATRA and 13RA were found to have a more pronounced inhibitory effect on LFA-3 expression than did DEX, the U937 cells being the most sensitive and the KB cells the least affected. The KB cells also expressed the lowest relative levels of drug receptors. DISCUSSION: The results showed that the three drugs differentially down-regulated LFA-3 expression by each of the cell lines. Moreover, the relative inhibitory effects of the drugs appeared to be related to the relative expression of the specific drug receptors by each of the cells. Our findings suggest that the down-regulation of LFA-3 in vitro might explain at least partly the efficacy of glucocorticoids and retinoids in the treatment of inflammatory diseases in vivo.     

抗氧化剂对系膜细胞醛糖还原酶活性的影响

目的：观察抗氧化剂对高糖培养下肾小球系膜细胞(MC)内醛糖还原酶(AR)活性的影响，探讨抗氧化剂治疗糖尿病并发症的分子机制。方法:将牛肾小球系膜细胞培养于含高糖的培养基中3周，同时加入抗氧化剂过氧化物酶和DMSO，采用间接法观察抗氧化剂对细胞内醛糖还原酶活性的影响结果:高糖作用下系膜细胞内山梨醇含量增加，表明醛糖还原酶活性增高，而抗氧化剂能抑制醛糖还原酶活性的增高。结论:氧化应激和多元醇通路关系密切，抗氧化剂通过降低AR的活性和清除多元醇通路中产生的氧自由基而对糖尿病并发症有治疗作用，为临床运用尤其是早期运用抗氧化剂治疗糖尿病提供了理论依据。     

3-Deoxyglucosone: metabolism, analysis, biological activity, and clinical implication.

3-Deoxyglucosone (3-DG) is synthesized via the Maillard reaction and the polyol pathway, and is detoxified to 3-deoxyfructose and 2-keto-3-deoxygluconic acid. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, N'-(carboxymethyl)lysine and pentosidine, among which imidazolone is the AGE most specific for 3-DG. As demonstrated by using gas chromatography-mass spectrometry or high-performance liquid chromatography, plasma 3-DG levels are markedly increased in diabetes and uremia. Although the plasma 3-DG levels had been controversial, it was clearly demonstrated that its plasma level depends on the deproteinization method by which either free or total 3-DG, presumably bound to proteins, is measured. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway, and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG, which may be due to the loss of 3-DG reductase activity in the end-stage kidneys, may lead to high plasma 3-DG level. The elevated 3-DG levels in plasma and erythrocytes may promote the formation of AGEs such as imidazolone, as demonstrated by immunohistochemistry and immunochemistry using an anti-imidazolone antibody. Although AGE-modified proteins prepared in vitro exhibit a variety of biological activities, known AGE structures have not yet been demonstrated to show any biological activities. Because 3-DG is potent in the formation of AGEs and has some biological activities, such as cellular toxicity, it may be more important in the development of diabetic and uremic complications than the known AGE structures. By demonstrating that treatment with an aldose reductase inhibitor reduces the erythrocyte levels of 3-DG and AGEs, such as imidazolone, light is shed on the mystery of how aldose reductase inhibitors may prove beneficial in diabetic complications. These evidences suggest that 3-DG plays a principal role in the development of diabetic and uremic complications.     

Optical method for real-time monitoring of drug concentrations facilitates the development of novel methods for drug delivery to brain tissue

The understanding of drug delivery to organs, such as the brain, has been hampered by the inability to measure tissue drug concentrations in real time. We report an application of an optical spectroscopy technique that monitors in vivo the real-time drug concentrations in small volumes of brain tissue. This method will facilitate development of new protocols for delivery of drugs to treat brain cancers. The delivery of many anticancer drugs to the brain is limited by the presence of the blood-brain barrier (BBB). Mitoxantrone (MTX) is a water-soluble anticancer drug that poorly penetrates the BBB. It is preliminarily determined in an animal model that the brain tissue uptake of chemotherapy agents-in this demonstration, MTX-delivered intra-arterially is enhanced when the BBB is disrupted.     

Thienocinnolinone alkanoic acid derivatives as aldose reductase inhibitors

A new series of 8-halogen-4,4a,5,6-tetrahydrothieno[2,3-h]cinnolinone-N2-alkanoic acids was prepared and tested for aldose reductase (ALR2) inhibitory activities. These compounds showed significant inhibitory activity against bovine lens ALR2, with the best compound 2e showing an IC(50) value of 31.4 microM. The presence of the C8-substituents here studied (Cl, Br) on the thienocinnolinone scaffold caused a decrease of the inhibitory potency by a factor of about 4 with respect to the unsubstituted parent compound, while the presence of a C8-methyl group, considered in a previous paper decreased the activity by a factor of about 2. Moreover, the length of the N2 alkanoic chain influences strongly the enzyme inhibitory activity. While most of the carboxylic acids ALR2 inhibitors are acetic acid derivatives, in the case of thienocinnolinone compounds, homologues higher than acetic acids showed to be more active.     

Parasite-specific trypanothione reductase as a drug target molecule

Trypanosomatids are the causative agents of African sleeping sickness, Chagas' disease, and the different manifestations of leishmaniasis. New drugs against these parasitic protozoa are urgently needed since the current drugs are unsatisfactory, in particular due to serious adverse side effects. In trypanosomes and leishmanias, the nearly ubiquitous glutathione/glutathione reductase system is replaced by trypanothione and trypanothione reductase. The essential role of trypanothione reductase in the parasite thiol metabolism and its absence from the mammalian host render the enzyme a highly attractive target molecule for a structure-based drug development against trypanosomatids. This article provides an overview on the known classes of trypanothione reductase inhibitors and their in vitro activity against parasitic protozoa. The (dis)advantages of the different types of compounds as potential drug candidates as well as modern computer-based approaches to the identification of new leads are discussed.     

Purification and enzymatic activity of an NADH-fumarate reductase and other mitochondrial activities of Leishmania parasites

A 65 kD membrane-associated NADH-fumarate reductase subunit, which has a molecular weight similar to that of one of the enzyme subunits from bacteria, was purified from Leishmania donovani promastigotes. NADH-fumarate reductase and other mitochondrial enzymatic activities of L. major and L. donovani promastigotes and amastigotes were investigated. The presence of NADH-fumarate reductase was demonstrated in digitonin-permeabilized L. major promastigotes and mitochondria of L. major and L. donovani promastigotes and amastigotes. The activity of solubilized NADH-fumarate reductase was measured in L. major and L. donovani promastigotes. Succinate exhibited a clear concentration-dependent inhibitory effect on fumarate reductase, whereas fumarate also exhibited a clear concentration-dependent inhibitory effect on succinate dehydrogenase. The data indicate that fumarate reductase is an obligatory component of the respiratory chain of the parasite. Since the enzyme is an important component in the intermediate metabolism in the Leishmania parasite and is absent in mammalian cells, it could be a potential target for antileishmanial drugs.     

Drug-induced changes in GABA content of nerve endings in 11 rat brain regions. Correlation to pharmacological effects

For determination of in vivo drug effects on nerve terminal GABA, a procedure was used which allowed the simultaneous measurement of GABA in nerve endings (synaptosomes) from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Postmortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. In vivo experiments with the GABA-elevating drugs aminooxyacetic acid (30 mg/kg i.p.) and valproic acid (200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosomal GABA levels in different brain regions. A comparison of these biochemical effects with anticonvulsant effects of both drugs in different seizure models supports the concept that GABA synapses in midbrain areas are critically involved in the control of seizure propagation.     

Bioinformatics analysis of diabetic retinopathy using functional protein sequences

Diabetic retinopathy is the leading cause of blindness among patients with diabetes mellitus. We evaluated the role of several proteins that are likely to be involved in diabetic retinopathy by employing multiple sequence alignment using ClustalW tool and constructed a phylogram tree using functional protein sequences extracted from NCBI. Phylogram was constructed using Neighbor-Joining Algorithm in bioinformatics approach. It was observed that aldose reductase and nitric oxide synthase are closely associated with diabetic retinopathy. It is likely that vascular endothelial growth factor, pro-inflammatory cytokines, advanced glycation end products, and adhesion molecules that also play a role in diabetic retinopathy may do so by modulating the activities of aldose reductase and nitric oxide synthase. These results imply that methods designed to normalize aldose reductase and nitric oxide synthase activities could be of significant benefit in the prevention and treatment of diabetic retinopathy.     

Synthesis of 3H-acetylcholine in the rabbit lacrimal gland and its release by electrical field stimulation.

Electrical field stimulation was applied to the rabbit lacrimal gland during incubation in vitro with 3H-choline, after which the efflux of tritiated metabolites was studied. In the presence of physostigmine 3H-acetylcholine was released into the incubation medium when the gland was stimulated electrically. The identity of the 3H-acetylcholine synthesized by the preparation was established by its identical ion exchange chromatographic properties to native acetylcholine. The tissue contents of 3H-acetylcholine and 3H-choline were 14.4 and 3.6%, respectively, of the total activities found in the gland. This study demonstrates that the rabbit lacrimal gland is innervated cholinergically. It is suggested that the method developed might be of value for studying effects of drugs on cholinergic neurotransmission in this organ.     

Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and sepiapterin reductase deficiency

DOPA responsive dystonia (DRD) and sepiapterin reductase (SR) deficiency are inherited disorders of tetrahydrobiopterin (BH4) metabolism characterized by the signs and symptoms related to monoamine neurotransmitter deficiency. In contrast to classical forms of BH4 deficiency DRD and SR deficiency present without hyperphenylalaninemia and thus cannot be detected by the neonatal screening for phenylketonuria (PKU). While DRD is mostly caused by autosomal dominant mutations in the GTP cyclohydrolase I gene (GCH1), SR deficiency is an autosomal recessive disease. The most important biochemical investigations for the diagnosis of these neurological diseases includes CSF investigations for neurotransmitter metabolites and pterins as well as neopterin and biopterin production in cytokine-stimulated fibroblasts. Discovery of SR deficiency opened new insights into alternative pathways of the cofactor BH4 via carbonyl, aldose, and dihydrofolate reductases. As a consequence of the low dihydrofolate reductase activity in the brain, dihydrobiopterin intermediate accumulates and inhibits tyrosine and tryptophan hydroxylases and uncouples nitric oxide synthase (nNOS), leading to neurotransmitter deficiency and possibly also to neuronal cell death.     

Symptomatic and neuroprotective properties of the aliphatic propargylamines

This short review describes the development of several families of aliphatic propargylamine drugs. The parent family--the aliphatic N-methylpropargylamines are potent, specific, irreversible MAO-B inhibitors, like R-deprenyl; but devoid of R-deprenyl's toxicity and amphetaminergic effects. In addition the parent drugs are potent antiapoptoic agents. Their desmethyl and further oxidized metabolites whilst devoid of MAO-B inhibitory properties of R-2HMP (our chosen drug candidate) are described along with the suggestion that this drug be developed for the treatment of several human neurodegenerative conditions.     

Effect of ingested pentoxifylline on neutrophil superoxide anion production.

Superoxide and other oxygen radicals produced by activated polymorphonuclear leukocytes (PMN) may be important causes of tissue damage in a number of inflammatory conditions. Therefore, a drug which suppresses PMN responses in vivo is potentially important. In vitro, pentoxifylline (PTOX) inhibits superoxide anion production when PMN are stimulated with an activated complement component (C5a Des Arg) or formyl peptides but only at concentrations not achieved in the circulation. The aim of this study was to determine whether PTOX has an effect on PMN responses in vivo. Superoxide anion production, monitored by lucigenin-enhanced chemiluminescence, was inhibited by 40.5% +/- 8.0% (n = 8, P < 0.009) for C5a Des Arg and 47.7% +/- 9.6% (n = 8, P < 0.009) for formyl-methionylleucylphenylalanine stimulation 1.5 h after ingestion of 400 mg of PTOX in a slow-release tablet, with some inhibitory effects persisting at 5 h. There was a strong correlation between reduced PMN response to activated complement and plasma concentrations of three PTOX metabolites (P < 0.05), but not with plasma concentrations of the parent drug. In vitro investigations with each of the four methylxanthines showed two of these metabolites to be most effective at reducing PMN respiratory burst activity, lactoferrin release, and the expression of CD11b and CD18 molecules. Furthermore, this in vitro inhibitory activity was achieved at concentrations of metabolites achievable in vivo. The results suggest that PTOX reduces oxygen radical production and protects against unwanted tissue damage in vivo by the action of its metabolites.     

Comparison in rats of the anti-inflammatory and gastric irritant effects of etodolac1 with several clinically effective anti-inflammatory drugs

The anti-inflammatory and gastric effects of etodolac were compared in the rat with those of seven clinically established nonsteroidal anti-inflammatory drugs. The anti-inflammatory potency of etodolac was found to lie between that of sulindac and piroxicam. Etodolac was 2.8 times more active than sulindac and 2.2 times less active than piroxicam. Compared to phenylbutazone it was 12.5 times more potent. The irritation produced on the gastric mucosa was less than that of sulindac, although this difference did not reach statistical significance. However, etodolac was significantly (p<0.05) less irritant than piroxicam. Of the drugs studied, etodolac showed the highest ratio between the irritant ED₅₀ and the dose which inhibited inflammation by 50%. From these results etodolac is predicted to be a potent anti-inflammatory drug with a high gastric tolerance. Clinical trials appear to confirm these predictions.     

Non-immunologic effects of cyclosporins

Cyclosporins are a group of molecules produced by fungi. The main cyclosporin, cyclosporin A, is widely known for its immunologic properties which have made it the leading drug for prophylactic therapy of transplant rejection. However, cyclosporin A (as well as several of its analogs with or without immunosuppressive properties) has a broader and more varied spectrum of biologic effects both in vivo and in vitro; renal, pancreatic, endothelial, muscle and nerve cells, hepatocytes, keratinocytes, and some microorganisms (plasmodium, schistosoma) are target cells whose metabolism or growth is affected by cyclosporins. Some of these activities are responsible for adverse effects but others may warrant the use of selected cyclosporins in clinical situations other than organ transplants.     

Novel neuroprotective anti-Alzheimer drugs with anti-depressant activity derived from the anti-Parkinson drug,rasagiline

A number of studies have shown that the selective monoamine oxidase (MAO)-B inhibitor l-selegiline has neuroprotective activities in several cell culture systems and in vivo. The suggestion has been made that the propargyl moiety in this molecule may have some intrinsic neuroprotective activity not related to its ability to bind covalently to MAO B and inhibit it. We have therefore developed a number of novel drugs based on rasagiline (N-propargyl-1R-(+)-aminoindan), a potent anti-Parkinson-propargyl-containing MAO-B inhibitor drug with structural resemblance to selegiline, for the treatment of Alzheimer's disease. These drugs possess a carbamate moiety for cholinesterase (ChE), and a propargyl group for MAO inhibition. The R-enantiomer of these compounds (TV3326) has ChE and MAO inhibitory activities in vivo and retains the neuroprotective properties of rasagiline. It also exhibits anti-depressant activity in animal models. The S-enantiomer does not inhibit MAO and has no anti-depressant activity, but it has similar ChE inhibitory and neuroprotective activities. Thus MAO inhibition by propargylamines is not a pre-requisite for neuroprotection. Rather, propargylamines have some intrinsic neuroprotective property whose mechanism of action requires further elucidation.