Glycerol monolaurate improved intestinal barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis in large yellow croaker (Larimichthys crocea) fed with high soybean oil diets.

Glycerol monolaurate (GML) is a potential candidate for regulating metabolic syndrome and inflammatory response. However, the role of GML in modulating intestinal health in fish has not been well determined. In this study, a 70-d feeding trial was conducted to evaluate the effect of GML on intestinal barrier, antioxidant capacity, inflammatory response and microbiota community of large yellow croaker (13.05 ± 0.09 g) fed with high level soybean oil (SO) diets. Two basic diets with fish oil (FO) or SO were formulated. Based on the SO group diet, three different levels of GML 0.02% (SO0.02), 0.04% (SO0.04) and 0.08% (SO0.08) were supplemented respectively. Results showed that intestinal villus height and perimeter ratio were increased in SO0.04 treatment compared with the SO group. The mRNA expressions of intestinal physical barrier-related gene odc and claudin-11 were significantly up-regulated in different addition of GML treatments compared with the SO group. Fish fed SO diet with 0.04% GML addition showed higher activities of acid phosphatase and lysozyme compared with the SO group. The content of malonaldehyde was significantly decreased and activities of catalase and superoxide dismutase were significantly increased in 0.02% and 0.04% GML groups compared with those in the SO group. The mRNA transcriptional levels of inflammatory response-related genes (il-1ß, il-6, tnf-α and cox-2) in 0.04% GML treatment were notably lower than those in the SO group. Meanwhile, sequencing analysis of bacterial 16S rRNA V4-V5 region showed that GML addition changed gut microbiota structure and increased alpha diversity of large yellow croaker fed diets with a high level of SO. The correlation analysis results indicated that the change of intestinal microbiota relative abundance strongly correlated with intestinal health indexes. In conclusion, these results demonstrated that 0.02%-0.04% GML addition could improve intestinal morphology, physical barrier, antioxidant capacity, inflammatory response and microbiota dysbiosis of large yellow croaker fed diets with a high percentage of SO.

The SWGEDWGEIW from Soybean Peptides Reduces Insulin Resistance in 3T3-L1 Adipocytes by Activating p-Akt/GLUT4 Signaling Pathway.

Diabetes mellitus, a group of metabolic disorders characterized by persistent hyperglycemia, affects millions of people worldwide and is on the rise. Dietary proteins, from a wide range of food sources, are rich in bioactive peptides with anti-diabetic properties. Notably, the protective mechanism of the single peptide SWGEDWGEIW (TSP) from soybean peptides (SBPs) on insulin resistance of adipocytes in an inflammatory state was investigated by detecting the lipolysis and glucose absorption and utilization of adipocytes. The results showed that different concentrations of TSP (5, 10, 20 µg/mL) intervention can reduce 3T3-L1 adipocytes' insulin resistance induced by inflammatory factors in a dose-dependent manner and increase glucose utilization by 34.2 ± 4.6%, 74.5 ± 5.2%, and 86.7 ± 6.1%, respectively. Thus, TSP can significantly alleviate the lipolysis of adipocytes caused by inflammatory factors. Further mechanism analysis found that inflammatory factors significantly reduced the phosphorylation (p-Akt) of Akt, two critical proteins of glucose metabolism in adipocytes, and the expression of GLUT4 protein downstream, resulting in impaired glucose utilization, while TSP intervention significantly increased the expression of these two proteins. After pretreatment of adipocytes with PI3K inhibitor (LY294002), TSP failed to reduce the inhibition of p-Akt and GLUT4 expression in adipocytes. Meanwhile, the corresponding significant decrease in glucose absorption and the increase in the fat decomposition of adipocytes indicated that TSP reduced 3T3-L1 adipocytes' insulin resistance by specifically activating the p-Akt/GLUT4 signal pathway. Therefore, TSP has the potential to prevent obesity-induced adipose inflammation and insulin resistance.

Effects of Tributyrin Supplementation on Growth Performance, Intestinal Digestive Enzyme Activity, Antioxidant Capacity, and Inflammation-Related Gene Expression of Large Yellow Croaker (Larimichthys crocea) Fed with a High Level of Clostridium autoethanogenum Protein.

An 8-week growth experiment was conducted to investigate effects of tributyrin (TB) supplementation on growth performance, intestinal digestive enzyme activity, antioxidant capacity, and inflammation-related gene expression of juvenile large yellow croaker (Larimichthys crocea) (initial weight of 12.90 ± 0.02 g) fed diets with high level of Clostridium autoethanogenum protein (CAP). In the negative control diet, 40% fish meal was used as the major source of protein (named as FM), while 45% fish meal protein of FM was substituted with CAP (named as FC) to form a positive control diet. Based on the FC diet, grade levels of 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% tributyrin were added to formulate other five experimental diets. Results showed that fish fed diets with high levels of CAP significantly decreased the weight gain rate (WGR) and specific growth rate (SGR) compared with fish fed the FM diet (P < 0.05). WGR and SGR were significantly higher than in fish fed diets with 0.05% and 0.1% tributyrin that fed the FC diet (P < 0.05). Supplementation of 0.1% tributyrin significantly elevated fish intestinal lipase and protease activities compared to FM and FC diets (P < 0.05). Meanwhile, compared to fish fed the FC diet, fish fed diets with 0.05% and 0.1% tributyrin showed remarkably higher intestinal total antioxidant capacity (T-AOC). Malondialdehyde (MDA) content in the intestine of fish fed diets with 0.05%-0.4% tributyrin was remarkably lower than those in the fish fed the FC diet (P < 0.05). The mRNA expressions of tumor necrosis factor α (tnfα), interleukin-1ß (il-1ß), interleukin-6 (il-6), and interferon γ (ifnγ) were significantly downregulated in fish fed diets with 0.05%-0.2% tributyrin, and the mRNA expression of il-10 was significantly upregulated in fish fed the 0.2% tributyrin diet (P < 0.05). In regard to antioxidant genes, as the supplementation of tributyrin increased from 0.05% to 0.8%, the mRNA expression of nuclear factor erythroid 2-related factor 2 (nrf2) demonstrated a trend of first rising and then decreasing. However, the mRNA expression of Kelch-like ECH-associated protein 1 (keap1) was remarkably lower in fish fed the FC diet than that fed diets with tributyrin supplementation (P < 0.05). Overall, fish fed tributyrin supplementation diets can ameliorate the negative effects induced by high proportion of CAP in diets, with an appropriate supplementation of 0.1%.

Myo-inositol improves growth performance and regulates lipid metabolism of juvenile Chinese mitten crab (Eriocheir sinensis) fed different percentage of lipid.

This study evaluated the effects of dietary myo-inositol (MI) on growth performance, antioxidant status and lipid metabolism of juvenile Chinese mitten crab (Eriocheir sinensis) fed different percentage of lipid. Crabs (4·58 (sem 0·05) g) were fed four diets including a normal lipid diet (N, containing 7 % lipid and 0 mg/kg MI), N with MI supplementation (N + MI, containing 7 % lipid and 1600 mg/kg MI), a high lipid diet (H, containing 13 % lipid and 0 mg/kg MI) and H with MI supplementation (H + MI, containing 13 % lipid and 1600 mg/kg MI) for 8 weeks. The H + MI group showed higher weight gain and specific growth rate than those in the H group. The dietary MI could improve the lipid accumulations in the whole body, hepatopancreas and muscle as a result of feeding on the high dietary lipid (13 %) in crabs. Besides, the crabs fed the H + MI diets increased the activities of antioxidant enzymes but reduced the malondialdehyde content in hepatopancreas compared with those fed the H diets. Moreover, dietary MI enhanced the expression of genes involved in lipid oxidation and exportation, yet reduced lipid absorption and synthesis genes expression in the hepatopancreas of crabs fed the H diet, which might be related to the activation of inositol 1,4,5-trisphosphate receptor (IP3R)/calmodulin-dependent protein kinase kinase-ß (CaMKKß)/adenosine 5'-monophosphate-activated protein kinase (AMPK) signalling pathway. This study demonstrates that MI could increase lipid utilisation and reduce lipid deposition in the hepatopancreas of E. sinensis fed a high lipid diet through IP3R/CaMKKß/AMPK activation. This work provides new insights into the function of MI in the diet of crustaceans.

Combined effects of polystyrene microplastics and copper on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia (Oreochromis niloticus).

Microplastics (MPs) coexist with other pollutants (such as heavy metals) in water, adversely impacting aquatic organisms, which might cause unpredictable ecological risks. This study aims to evaluate the effect of copper (Cu2+) and polystyrene microplastics (PS-MPs) on antioxidant capacity, immune response and intestinal microbiota of Nile tilapia. Cu2+ and PS-MPs co-exposure enhanced Cu2+ bioaccumulation in the liver of fish compared with Cu2+-alone exposure. Fish exposed to PS-MPs and Cu2+ displayed histopathologic alterations in the liver, intestine and gill. Exposure at low concentrations of Cu2+ in the C0 and CP0 groups can improve antioxidant capacity and immune response, while oxidative damage and inflammation existed in the high concentration of Cu2+ groups. Intestinal microbiota results showed that the diversity and structure were changed by Cu2+ and PS-MPs exposure, and harmful bacterium even increased at high concentration of Cu2+ and PS-MPs exposure groups. All in all, PS-MPs aggravate the accumulation of Cu2+ and lead to perturbations in biological systems of Nile tilapia.

Impact of Dietary Vitamin D3 Supplementation on Growth, Molting, Antioxidant Capability, and Immunity of Juvenile Chinese Mitten Crabs (Eriocheir sinensis) by Metabolites and Vitamin D Receptor.

Vitamin D3 (vit-D3), as an indispensable and fat-soluble nutrient, is associated with skeletal mineralization and health in mammals. However, such associations have not been well studied in economically important crustaceans. Six levels of vit-D3 with isonitrogenous and isolipidic diets were used to feed Eriocheir sinensis. The range of optimal vit-D3 requirements is 5685.43-10,000 IU/kg based on growth. The crabs fed 9000 IU/kg vit-D3 showed the best growth performance. This vit-D3 dose significantly increased antioxidant capacity in the hepatopancreas and intestine and was optimal for molting and innate immunity via quantitative polymerase chain reaction analysis. Transcriptomics analyses indicate that vit-D3 could alter protein processing in the endoplasmic reticulum, steroid biosynthesis, and antigen processing and presentation. As shown by the enzyme-linked immunosorbent assay, vit-D3 could improve vitamin D receptor, retinoic acid receptor, and C-type lectins concentrations. The 1α,25-dihydroxy vit-D3 content in serum was significantly higher in 3000-9000 IU/kg vit-D3. The study suggests that dietary vit-D3 and its metabolites can regulate molting and innate immunity in crabs.

Influences of dietary vitamin D3 on growth, antioxidant capacity, immunity and molting of Chinese mitten crab (Eriocheir sinensis) larvae.

This study investigates the effects of vitamin D3 (VD3) on growth performance, antioxidant capacity, immunity and molting of larval Chinese mitten crab Eriocheir sinensis. A total of 6,000 larvae (7.52 ± 0.10 mg) were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary VD3 (0, 3000, 6000, 9000, 12000 and 36000 IU/kg) respectively for 23 days. The highest survival and molting frequency were found in crabs fed 6000 IU/kg VD3. Weight gain, specific growth rate, and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg VD3 compared to the control. Broken-line analysis of molting frequency, weight gain and specific growth rate against dietary VD3 levels indicates that the optimal VD3 requirement for larval crabs is 4825-5918 IU/kg. The highest whole-body VD3 content occurred in the 12000 IU/kg VD3 group, and the 25-dihydroxy VD3 content decreased with the increase of dietary VD3. The malonaldehyde content was lower than the control. Moreover, the superoxide dismutase activity, glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg VD3 were significantly higher than in control. Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress, and the relative mRNA expressions indicate vitamin D receptor (VDR) is the important regulatory element in molting and innate immunity. The molting-related gene expressions showed that the response of crab to salinity was self-protective. This study would contribute to a new understanding of the molecular basis underlying molting and innate immunity regulation by vitamin D3 in E. sinensis.

Effects of dietary T-2 toxin on gut health and gut microbiota composition of the juvenile Chinese mitten crab (Eriocheir sinensis).

The current study aims to investigate the effects of dietary T-2 toxin on the intestinal health and microflora in the juvenile Chinese mitten crab (Eriocheir sinensis) with an initial weight 2.00 ± 0.05 g. Juvenile crabs were fed with experimental diets supplemented with T-2 toxin at 0 (control), 0.6 (T1 group), 2.5 (T2 group) and 5.0 (T3 group) mg/kg diet for 8 weeks. Dietary T-2 toxin increased the malondialdehyde (MDA) content and the expression of Kelch-like ECH-associated protein 1 (keap1) gene while the expression of cap 'n' collar isoform C (CncC) decreased in the intestine. The activities of glutathione peroxidase (GSH-Px) and total anti-oxidation capacity (T-AOC) in the intestine increased only in the lower dose of dietary T-2. Dietary T-2 toxin significantly increased the mRNA expression of caspase-3, caspase-8, Bax and mitogen-activated protein kinase (MAPK) genes and the ratio of Bax to Bcl-2 accompanied with a reduction of Bcl-2 expression. Furthermore, T-2 toxin decreased the mRNA levels of antimicrobial peptides (AMPs), peritrophic membrane (PM1 and PM2) and immune regulated nuclear transcription factors (Toll-like receptor: TLR, myeloid differentiation primary response gene 88: Myd88, relish and lipopolysaccharide-induced TNF-α factor: LITAF). The richness and diversity of the gut microbiota were also affected by dietary T-2 toxin in T3 group. The similar dominant phyla in the intestine of the Chinese mitten crab in the control and T3 groups were found including Bacteroidetes, Firmicutes, Tenericutes and Proteobacteria. Moreover, the inclusion of dietary T-2 toxin of 4.6 mg/kg significantly decreased the richness of Bacteroidetes and increased the richness of Firmicutes, Tenericutes and Proteobacteria in the intestine. At the genus level, Dysgonomonas and Romboutsia were more abundant in T3 group than those in the control. However, the abundances of Candidatus Bacilloplasma, Chryseobacterium and Streptococcus in T3 group were lower than those in the control. This study indicates that T-2 toxin could cause oxidative damage and immunosuppression, increase apoptosis and disturb composition of microbiota in the intestine of Chinese mitten crab.

T-2 toxin in the diet suppresses growth and induces immunotoxicity in juvenile Chinese mitten crab (Eriocheir sinensis).

The T-2 toxin is a trichothecene mycotoxin and is highly toxic to aquatic animals, but little is known on its toxic effect in crustaceans. In the present study, the crab juveniles were fed with diets containing four levels of T-2 toxin: 0 (control), 0.6 (T1), 2.5 (T2) and 5.0 (T3) mg/kg diet for 56 days to evaluate its impact on the juvenile of Chinese mitten crab (Eriocheir sinensis). The crabs fed the T-2 toxin diets had significantly lower weight gain and specific growth rate than those fed the control diet. Moreover, crab survival in T3 group was obviously lower than that in the control. Oxidative stress occurred in all the treatment groups as indicated by higher activities of total superoxide dismutase, glutathione peroxidase, and total antioxidant capacity than those in the control. The total hemocyte count, respiratory burst, phenoloxidase in the hemolymph, and phenoloxidase, acid phosphatase and alkaline phosphatase in the hepatopancreas of crabs fed T-2 toxin were significantly lower than those in the control. The transcriptional expressions of lipopolysaccharide-induced TNF-alpha factor, relish, and the apoptosis genes in the hepatopancreas were induced by dietary T-2 toxin. The genes related to detoxication including cytochrome P450 gene superfamily and glutathione S transferase were induced in low concentration, then decreased in high concentration. Dietary T-2 toxin damaged the hepatopancreas structure, especially as seen in the detached basal membrane of hepatopancreatic tubules. This study indicates that dietary T-2 toxin can reduce growth performance, deteriorate health status and cause hepatopancreas dysfunction in crabs.

Effect of single and combined immunostimulants on growth, anti-oxidation activity, non-specific immunity and resistance to Aeromonas hydrophila in Chinese mitten crab (Eriocheir sinensis).

This study evaluates the effect of dietary supplementation of immunostimulants on the Chinese mitten crab (Eriocheir sinensis) with a single administration of mannan oligosaccharide (MOS), or its combination with either ß-glucan or with inulin for 8 weeks. Four diets included an untreated control diet (C), MOS alone (3 g kg-1, M), MOS with ß-glucan (3 g kg -1 MOS + 1.5 g kg -1 ß-glucan, MB), and MOS with inulin (3 g kg -1 MOS + 10 g kg -1 inulin, MI). The weight gain and specific growth rate of the crabs fed M, MB, and MI diets were improved by lowing feed conversion ratio. The growth and feed utilization of the crabs fed the MB diet were improved compared with the other three groups. The crabs fed the M, MB and MI diets showed a higher intestinal trypsin activity than that in the M and control groups. The highest trypsin activity in the hepatopancreas was observed in the MB group. Crabs fed M, MB and MI diets increased antioxidant system-related enzyme activities, but reduced malondialdehyde. The highest activities of alkaline phosphatase, acid phosphatase, lysozyme and phenol oxidase in the gut and the respiratory burst of the crabs were found in the MB group. The MB diet promoted the mRNA expression of E. sinensis immune genes (ES-PT, ES-Relish, ES-LITAF, p38MAPK and Crustin) compared with the control. After 3 days of infection with Aeromonas hydrophila, the highest survival of crabs was also found in the MB group. This study indicates that the combination of MOS with ß-glucan or with inulin can improve growth, antioxidant capacity, non-specific immunity and disease resistance in E. sinensis.

Dietary Aroclor 1254-Induced Toxicity on Antioxidant Capacity, Immunity and Energy Metabolism in Chinese Mitten Crab Eriocheir sinensis: Amelioration by Vitamin A.

Effects of dietary Polychlorinated biphenyl (PCB) exposure and dietary vitamin A supplementation on Chinese mitten crab Eriocheir sinensis were studied with the aim to explain dietary PCB toxicity and toxic alleviation by vitamin A intake in crab. Four diets were used including three experimental diets containing 0, 80000 or 240000 IU/kg vitamin A with each experimental diet containing 10 mg PCB/kg diet, and a control diet (without vitamin A and PCB supplementation) in 56 days feeding trial. Crabs fed the PCB-only diet had significantly lower weight gain than those fed the control diet. No significant difference was observed in crab survival among all groups. Crabs fed the PCB-only diet had a significantly higher malondialdehyde content and antioxidase superoxide dismutase activity in the serum and hepatopancreas, and higher erythromycin N-demethylase and glutathione S-transferase activities in the hepatopancreas than those fed the control diet. However, supplementation of dietary vitamin A decreased the levels of all these parameters. The hepatopancreatic cytochrome P450 2 and 4 (CYP2, CYP4), fatty acid binding proteins 3 and 10 (FABP3, FABP10) and intracellular lipolytic enzyme (IL) Messenger Ribonucleic Acid (mRNA) levels in the PCB-only group were significantly higher than those in the control group, and dietary 240000 IU/kg vitamin A supplementation decreased hepatopancreatic CYP4, FABP3, FABP10 and IL enzyme mRNA level. The crabs fed 80000 IU/kg vitamin A supplementation diet had the highest level of retinoid X receptor mRNA in the hepatopancreas. The structure of the hepatopancreas was damaged and the deposit of lipid droplets decreased with dietary PCB exposure. Both levels of vitamin A supplementation alleviated the damage and increased lipid droplets in the hepatopancreas. Dietary PCB exposure significantly reduced total hemocyte count (THC), and phenoloxidase, acid phosphatase activities in the serum. Post-challenge survival of crab in the experimental PCB-only diet group was low compared with that in the control. Supplementation of 240000 IU/kg vitamin A significantly increased the THC and phenoloxidase activity in the serum and post-challenge survival compared with those in the PCB-only group. This study indicates that dietary vitamin A can improve the antioxidant capacity, immune response, detoxification enzymes activities, energy metabolism and hepatopancreas tissue structure of Chinese mitten crab fed PCB contaminated diets.

Dietary yeast culture modulates immune response related to TLR2-MyD88-NF-kß signaling pathway, antioxidant capability and disease resistance against Aeromonas hydrophila for Ussuri catfish (Pseudobagrus ussuriensis).

The aim of the present study was to investigate effects of dietary yeast culture on immune response related to TLR2-MyD88-NF-kß signaling pathway, antioxidant capability and disease resistance against Aeromonas hydrophila for Ussuri catfish (Pseudobagrus ussuriensis). A total of 240 Ussuri catfish (mean weight of 7.39 ±â€¯0.32 g) were randomly distributed into four groups that fed diets containing 0 (Y0), 10 (Y1), 20 (Y2) and 30 (Y3) g kg-1 yeast culture for 8 weeks. The results indicated that dietary 10 g kg-1 yeast culture supplementation significantly down-regulated mRNA levels of TLR2, MyD88, NF-kß p65, IL-1ß and IL-8 in the liver tissue compared with the control group (P < 0.05). Simultaneously, serum lysozyme (LZM) activity, respiratory burst activity (RBA) of phagocytes, plasma alkaline phosphatase (AKP) activity and immunoglobulin M (IgM) content were significantly improved in fish fed Y1 diet (P < 0.05). Fish fed Y1 diet had significantly higher serum alternative complement pathway activity (ACH50) and plasma complement 3 (C3) content than the Y3 group (P < 0.05). However, no significant differences were observed in plasma acid phosphatase (ACP) activity and complement 4 (C4) content among the groups (P > 0.05). Fish cumulative mortality rate (CMR) in the Y1 and Y2 groups were significantly lower than that in Y0 and Y3 groups (P < 0.05), and the lowest CMR was observed in the Y1 group after challenge by A. hydrophila. The highest hepatic superoxide dismutase and glutathione peroxidase activities, total antioxidant capacity and the lowest malondialdehyde content were found in Y1 group, but no significant difference was found in hepatic catalase activity among the groups (P > 0.05). These results demonstrate that dietary 10 g kg-1 yeast culture could effectively improve the immunity, antioxidant capability and disease resistance against A. hydrophila for Ussuri catfish and could down-regulate the mRNA expression levels of pro-inflammatory cytokines modulated by TLR2-MyD88-NF-kß signaling pathway.

Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs).

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

Discovery of CYT997: a structurally novel orally active microtubule targeting agent.

CYT997 was discovered as a potent tubulin polymerization inhibitor possessing potent cytotoxic activity against a range of cancer cells. Details of SAR studies, pharmacokinetic investigations and synthesis of compounds leading to the discovery of CYT997 are reported.

Discovery of 2-(alpha-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Pyrido-pyrimidine derivative CYC10424 inhibits glycosaminoglycan changes on vascular smooth muscle-derived proteoglycans and reduces lipoprotein binding.

Cardiovascular disease is a major cause of mortality with the underlying process being atherosclerosis. Modified proteoglycans bind low-density lipoproteins (LDL), a critical initial step in the atherosclerotic cascade, representing a potential therapeutic target. Platelet-derived growth factor (PDGF) stimulates proteoglycan synthesis and is strongly implicated in atherogenesis. In human vascular smooth muscle cells (VSMCs), CYC10424 (Cytopia Research Ltd), a pyrido-pyrimidine derivative, dose-dependently decreased PDGF-mediated radiolabel incorporation into proteoglycans associated with an increase in electrophoretic mobility by SDS-PAGE. PDGF stimulated increases in both chemically-cleaved and xyloside-associated glycosaminoglycan (GAG) chain size, which were inhibited in the presence of CYC10424 by size exclusion chromatography (Sepharose CL-6B). CYC10424 treatment inhibited the PDGF effect to increase the 6:4 position sulfation ratio of monosulfated disaccharides by fluorophore-assisted carbohydrate electrophoresis. Proteoglycans derived from cells treated with CYC10424 had a decreased binding affinity and capacity to human LDL by gel mobility shift assay. CYC10424 and related compounds are possible candidates as therapeutic agents for the prevention of lipid deposition as characteristic of diseases such as atherosclerosis.

DNA threading bis(9-aminoacridine-4-carboxamides): effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest.

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC(50) values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.

Synthesis and cytotoxic activity of N-[(alkylamino)alkyl]carboxamide derivatives of 7-oxo-7H-benz[de]anthracene, 7-oxo-7H-naphtho[1,2,3-de]quinoline, and 7-oxo-7H-benzo[e]perimidine.

7-Oxo-7H-naphtho[1,2,3-de]quinoline-11-carboxamides and analogues were prepared and evaluated for in vitro and in vivo antitumor activity. Chromophore variations included 'deaza' (7-oxo-7H-benz[de]anthracene) and 'diaza' (7-oxo-7H-benzo[e]perimidine) analogues, and side chain variations included chiral alpha-methyl compounds. The naphthoquinolines were the most cytotoxic, with IC(50) values of 5-20 nM, and showed the strongest DNA binding, with high selectivity for G-C rich DNA. The chiral alpha-methyl analogues were 10-20-fold more cytotoxic than the parent des-methyl compound. Both enantiomers provided substantial growth delays against s.c. colon 38 tumors in mice, with the R-enantiomer more active than the S (tumor growth delays of >35 and 12 days, respectively).

Bisintercalating threading diacridines: relationships between DNA binding, cytotoxicity, and cell cycle arrest.

We have synthesized a series of bis(9-aminoacridine-4-carboxamides) linked via the 9-position with neutral flexible alkyl chains, charged flexible polyamine chains, and a semirigid charged piperazine-containing chain. The carboxamide side chains comprise N,N-dimethylaminoethyl and ethylmorpholino groups. The compounds are designed to bisintercalate into DNA by a threading mode, in which the side chains are intended to form hydrogen-bonding contacts with the O6/N7 atoms of guanine in the major groove, and the linkers are intended to lie in the minor groove. By this means, we anticipate that they will dissociate slowly from DNA, and be cytotoxic as a consequence of template inhibition of transcription. The dimers remove and reverse the supercoiling of closed circular DNA with helix unwinding angles ranging from 26 degrees to 46 degrees, confirming bifunctional intercalation in all cases, and the DNA complexes of representative members dissociate many orders of magnitude more slowly than simple aminoacridines. Cytotoxicity for human leukemic CCRF-CEM cells was determined, the most active agents having IC(50) values of 35-50 nM in a range extending over 20-fold, with neither the dimethylaminoethyl nor the ethylmorpholino series being intrinsically more toxic. In common with established transcription inhibitors, the morpholino series, with one exception, have no effect on cell cycle distribution in randomly dividing CCRF-CEM populations. By contrast, the dimethylaminoethyl series, with two exceptions, cause G2/M arrest in the manner of topoisomerase poisons, consistent with possible involvement of topoisomerases in their mode of action. Thus, the cellular response to these bisintercalating threading agents is complex and appears to be determined by both their side chain and linker structures. There are no simple relationships between structure, cytotoxicity, and cell cycle arrest, and the origins of this complexity are unclear given that the compounds bind to DNA by a common mechanism.