Antihyperglycaemic activity of 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal in diabetic mice

We have recently generated lipophilic D-xylose derivatives that increase the rate of glucose uptake in cultured skeletal muscle cells in an AMP-activated protein kinase (AMPK)-dependent manner. The derivative 2,4:3,5-dibenzylidene-D-xylose-diethyl dithioacetal (EH-36) stimulated the rate of glucose transport by increasing the abundance of glucose transporter-4 in the plasma membrane of cultured myotubes. The present study aimed at investigating potential antihyperglycaemic effects of EH-36 in animal models of diabetes. Two animal models were treated subcutaneously with EH-36: streptozotocin-induced diabetes in C57BL/6 mice (a model of insulin-deficient type 1 diabetes), and spontaneously diabetic KKAy mice (Kuo Kondo rats carrying the A(y) yellow obese gene; insulin-resistant type 2 diabetes). The in vivo biodistribution of glucose in control and treated mice was followed with the glucose analogue 2-deoxy-2-[(18) F]-D-glucose; the rate of glucose uptake in excised soleus muscles was measured with [(3) H]-2-deoxy-D-glucose. Pharmacokinetic parameters were determined by non-compartmental analysis of the in vivo data. The effective blood EH-36 concentration in treated animals was 2 μM. It reduced significantly the blood glucose levels in both types of diabetic mice and also corrected the typical compensatory hyperinsulinaemia of KKAy mice. EH-36 markedly increased glucose transport in vivo into skeletal muscle and heart, but not to adipose tissue. This stimulatory effect was mediated by Thr(172) -phosphorylation in AMPK. Biochemical tests in treated animals and acute toxicological examinations showed that EH-36 was well tolerated and not toxic to the mice. These findings indicate that EH-36 is a promising prototype molecule for the development of novel antidiabetic drugs.     

Garlic allyl derivatives interact with membrane lipids to modify the membrane fluidity

As a novel approach to the mode of medicinal action of garlic, its constituents were comparatively studied with respect to their interactions with membrane lipids to modify the membrane fluidity. Allyl derivatives rigidified tumor cell and platelet model membranes consisting of unsaturated phospholipids and cholesterol at 20–500 μM with the potency being diallyl trisulfide (DATS) > diallyl disulfide (DADS) by preferentially acting on the hydrocarbon cores of lipid bilayers. They were also effective in rigidifying candida cell model membranes prepared with ergosterol and phospholipids at 100–500 μM with the potency being DADS > DATS > diallyl sulfide (DAS), but not bacteria cell model membranes without ergosterol. Alliin, a precursor of these DASs, was not active on any membranes at 500 μM. Both relative intensity and selectivity in membrane effects correlated with those in antiproliferative, antiplatelet and antimicrobial effects. In cell culture experiments, membrane-active DASs inhibited the growth of tumor cells cultured for 24 and 48 h at 20–500 μM to show the potency being DATS > DADS, together with rigidifying cell membranes by acting on their deeper regions more intensively. However, membrane-inactive allyl derivatives were not growth-inhibitory on tumor cells. The membrane lipid interactions of DASs appear to be one of possible mechanisms underlying different effects of garlic.     

Synthesis of new N-isobutyryl-L-cysteine/MEA conjugates: evaluation of their free radical-scavenging activities and anti-HIV properties in human macrophages

Four novel N-isobutyryl-l-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test, and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro. The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.     

Design of Benzene-1,2-diamines as selective inducible nitric oxide synthase inhibitors: a combined de novo design and docking analysis

Selective inhibition of inducible nitric oxide synthases (iNOS) has been a challenging problem for researchers pursuing work in finding methods to treat inflammatory disorders, shock, etc. Though many inhibitors have been studied to date, all are associated with selectivity or potency problems. Additionally, most of the reported compounds have several similarities and fewer number of novel structures are being tried. There is an increasing need to design novel molecules for this target. In this work, de novo design using LUDI, combined with docking analysis using FlexX has been employed in an attempt to identify novel scaffolds. Benzene-1,2-diamines were identified which could mimic the interactions of the substrate analogs and other inhibitors. Comparative docking scores in each of the isoforms of nitric oxide synthase were employed to recognize hits for iNOS selectivity. Figure Figure shows the docked poses of the ligand M226 along with that of the reference GW274150. (FlexX analysis)     

XANES study of the carboxylate binding mode in two pterin hydroxylases

The 2-His-1-carboxylate triad is an Fe(II)-binding motif common to several enzyme families. Within the catalytic cycle, the metal ion seems to alter between hexa- and pentacoordination, providing an open space in the Fe moiety for dioxygen binding. Anyhow, based on crystallographic studies, the picture is not fully consistent as different coordination numbers are reported for similar states. Moreover, the orientation of the metal-coordinating carboxylate varies in these studies. These differences are reflected in the XANES spectra analyzed in this paper. For isolated tyrosine and phenylalanine hydroxylase, the different active-site structures postulated by protein crystallography and further improved using spectroscopic data result in calculated XANES pattern that resemble very well the experiments. This work shows that structural differences in the non-coordinated oxygen of the carboxylate have no effect in the EXAFS but cause a change in the white-line intensity that can be successfully modeled within the muffin-tin approximation in small clusters. Thus, the study shows a way to analyze the 'carboxylate shift'. This highlights the potential of XANES analysis and clearly shows that the mentioned structural differences are present in solution as well, and does neither reflect the crystallization artifacts nor result from radiation damage or lacking resolution.     

Resistance in hybrid derivatives of Lycopersicon accessions against leaf caterpillar, Spodoptera litura Fab.

Four tomato accessions, namely Ac 238, Roma, Seijima Jeisei and Varushanadu Local selected from preliminary screening of 321 accessions and their hybrid derivatives, namely HY₁F₁ (Varushanadu Local × Ac 238), HY₂F₁ (Varushanadu Local × Roma), HY₃F₁ (Ac 238 × Roma) (first generation), HY₁F₂, HY₂F₂ and HY₃F₂ (second generation) were evaluated for their resistance in comparison with a susceptible check, 1979 against the leaf caterpillar, Spodoptera litura Fab. both under field and laboratory conditions at Annamalainagar, Tamil Nadu, India. In the field evaluation, accession Seijima Jeisei and hybrid HY3F1 recorded the minimum larval population. In glasshouse and laboratory studies, Seijima Jeisei was the least preferred by S. litura for both feeding and oviposition. The hybrid HY₃F₁ exerted pronounced antibiosis effect on S. litura. Among the various biophysical and biochemical bases of resistance, contents of reducing sugar and phenol were found negatively correlated with S. litura larval feeding. Among the hybrids, HY₃F₁ and HY3F2 and the parent Seijima Jeisei recorded higher fruit yield per plant in both seasons. Considerable variation in the resistance traits of the hybrids was observed when compared with the parents.     

The effect of mildronate and related substances on levels of thyroid hormones and some intermediates of lipid and carbohydrate metabolism in hyperthyroid and hypothyroid rats

The effects of mildronate [3(2,2,2-trimethylhydrazinium) propionate dihydrate], γ-butyrobetaine (GBB) and their combination (neomildronate) on the level of thyroid hormones and some intermediates of basal metabolism (free fatty acids, triglycerides, glucose) in serum of laboratory rats with various dysfunctions of thyroid glands including idiopathic hyperfunction and also hypofunction induced by administration of 6-propyl-2-thiouracil (PTU) or L-carnitine administration. Intraperitoneal injections of mildronate (150 mg/kg) during 20 days to male Wistar rats with elevated level of thyroid hormones and basal metabolism normalized thyroxin level and parameters of lipid metabolism in serum. Administration of the compounds studied to rats with hypothyroidism induced by administration of PTU or L-carnitine did not influence natural recovery of the hormonal level. Possible biochemical role of these pharmacological treatments is discussed in terms of in regulation of thyroid gland function.     

Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro

Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of the parent CS. The minimum value of MIC and MBC of the derivatives against E. coli was 15.62 and 62.49 microg/mL, respectively. All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50-500 microg/mL; the maximum inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal activity; higher substitution resulted in stronger antifungal activity.     

The inhibitory effect of various indolyl amino acid derivatives on arginase activity in macrophages

Summary. Numerous indolyl amino acids and their derivatives inhibited arginase activity. The inhibition was found to be non-competitive, – at least partly – allosteric, and independent on manganese ions in the active site, and it cannot be explained by the dissociation of arginase homotrimers. Indole alone is weakly inhibitory; however, the presence of three-carbon side chains and their net charges is favorable for the inhibition. The binding of the inhibitory compounds caused only minor changes in the steric structure of arginase: a slight increase in α-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and β-turn sections. A slight alteration in the tertiary structure was also found using tryptophane fluorescence studies, but buried apolar side chains were not transposed to the protein surface. Computer studies that were performed did not provide additional structural information. Authors’ address: András Hrabák, Department of Medical Chemistry, Molecular Biology and Patho-biochemistry, Semmelweis University Medical School, Budapest, VIII. Puskin u. 9., H-1444 POB 260, Hungary     

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.