Discovery of new azaindole-based PI3Kα inhibitors: apoptotic and antiangiogenic effect on cancer cells

Phosphatidylinositol-3-kinase alpha (PI3Kα) is an important target in cancer due to the deregulation of the PI3K/AKT signaling pathway in many tumors. In this study, we designed [3,5-d]-7-azaindole analogs as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3,5-positions of azaindole, we developed the SAR (Structure-activity relationship) and identified a series of potent PI3Kα inhibitors. Representative azaindole derivatives showed activity in a cellular proliferation and apoptosis assays. Moreover, B3 exhibited strong antiangiogenic effects on cancer cells.     

Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.     

Bimodal effect of humic acids on the LPS-induced TNF-α release from differentiated U937 cells

Humic substances (HS) have been reported to possess anti-inflammatory as well as pro-inflammatory properties. The anti-inflammatory activity was demonstrated in the rat paw edema model and we found a preliminary explanation in the 5-lipoxygenase inhibitory effect of humic acids (HA). The pro-inflammatory activity is reflected by the production and release of pro-inflammatory cytokines in HA-treated neutrophilic granulocytes. With regard to the potential use of HA as antiviral and UV-protective agents it appears advisable to investigate the role of HS in the inflammation process in more detail. Hence we tested four different HS preparations – two naturally occurring HA from the Altteich peatland in Germany, one fulvic acid (FA) preparation from a Finnish spruce forest and a synthetic HA-like polymer (caffeic acid oxidation product, KOP) for their influence on the lipopolysaccharide (LPS)-induced TNF-α release in human U937 cells. In addition, the cytotoxicity of HS was determined.The results demonstrate a concentration-dependent bimodal effect of HA on the TNF-α release of differentiated LPS-stimulated U937 cells for both the natural black peat HA from the Altteich peatland and the HA-like polymer KOP. Low HA concentrations (10–80 μg/ml) enhanced the TNF-α release by up to threefold (pro-inflammatory activity), while HA concentrations >100 μg/ml reduced it about 10-fold (anti-inflammatory activity). FA failed to enhance TNF-α release, but reduced it at higher concentrations (>200 μg/ml) by the half. Brown water HA did not exert any significant effect on TNF-α release. No HS-stimulated TNF-α release was also observed in the absence of exogenously supplied LPS. This means that HS, unlike endotoxin, are no inflammation-causing agents for LPS-untreated cells. Differences in the effect of individual HS on TNF-α release are discussed in connection with the polyanionic character of HS, their molecular mass distribution and the hitherto imperfectly known chemical structure.     

The effect of anti-tumor necrosis factor (TNF)-α monoclonal antibody on allergic cutaneous late phase reaction in mice

Biphasic cutaneous reaction with peak response at 1 (early phase) and 24 to 48 hour (late phase)was elicited by epicutaneous challenge with antigen in actively and passively sensitized mice. Mice were actively immunized with dinitrophenylated (DNP) ascaris antigen and challenged with dinitrofluorobenzene (DNFB). Passively sensitization was carried out by the injection of monoclonal anti-DNP-IgE antibody into mice and challenge was elicited with DNFB. Prednisolone at doses of 3 to 10 mg/kg clearly inhibited both early and late phase reactions in either sensitized mice. Monoclonal anti-tumor necrosis factor (TNF)-α antibody inhibited the late phase cutaneous reaction in actively sensitized mice. Anti-interleukin-5 (IL-5) monoclonal antibody has no effect on both phase reactions in either actively and passively sensitized animals. These results indicate the possible participation of TNF-α in allergic cutaneous late phase reaction in actively sensitized mice.     

An inhibitor in the Willebrand-Jürgens syndrome and its effect on the factor VIII molecule properties]

In a patient with a clinically serious Willebrand-Jürgen's syndrome an inhibitor appeared at the age of 1 1/2 years, which, contrary to inhibitors in haemophilia A, was directed against all properties of factor VIII molecule. In a quantitative test the height of the inhibitor level to factor VIIIag was determined. Administrations of plasma concentrate resulted in a titre increase which could not be suppressed even by an immunosuppressive therapy with cyclophosphamide. After a long break in the substitution a severe bleeding could be successfully treated and the substitution effect for factor VIIIc, factor VIIIag and Ristocetin co-factor could be identified for several days.     

Central effects of TNFα on thermogenesis and fever in the rat

Intracerebroventricular (icv) injection of purified recombinant human tumour necrosis factor (TNF , 4–8g) in conscious rats, produced increases in colonic temperature (1.0°C) and resting oxygen consumption (VO₂, 14%) which were maximal after 80–90 minutes. Pretreatment with propranolol (10mg/kg s.c) significantly inhibited the rise in VO₂, and prevented the increase in body temperature. Icv injection of an antagonist to corticotropin releasing factor (-helical CRF 9-41, 25 g), which prevents the pyrogenic and thermogenic actions of interleukin-1, did not influence the effects of TNF on temperature or VO₂. Injection of a fragment of TNF (113–130 amino acid sequence) did not affect body temperature or VO₂. TNF injection (icv) significantly increased brown adipose tissue (BAT)in vitro mitochondrial GDP binding, and this effect was slightly inhibited, but not prevented, by surgical denervation of the tissue, and was unaffected by pretreatment with -helical CRF 9-41. These data indicate that TNF can stimulate thermogenesis by a direct central action. The effects are largely, but not totally, dependent on the sympathetic nervous system but, unlike the thermogenic actions of interleukin they do not require release of CRF.     

On the 48÷80-induced secretion of tissue mast cells and its mitogenic effect on nearby cells in the intact rat

Histamine release from tissue-bound mast cells and cell proliferation in the proper mesentery in the intact rat was quantitated following in intraperitoneal injection of graded doses of compound 48/80. The dose-response curves were sigmoid-like in linear-log plots. ED50 for histamine release was 0.035-0.040 and for increased cell proliferation 0.040-0.048 microgram per g BW. The proliferative response following mast-cell secretion ceased after a period of between 48-72 h, irrespective of whether a high or a low dose of 48/80 was used. Basal on the net rate of histamine synthesis (ca. 0.45 microgram/g mesentery wet weight/h) after an initial injection of 48/80, on the extent of histamine release and the proliferative response after a repeated injection of 48/80, it is concluded that there is a lag period of at least 3 days before proliferation can be re-stimulated by renewed 48/80-induced mast-cell secretion.     

Study on genetic variations of PPARα gene and its effects on thermal tolerance in Chinese Holstein

Peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. In this research, polymerase chain reaction (PCR) technique was used to amplify 766 and 589 bp fragments of intron 3 and 7 of PPARα gene in Chinese Holstein (n = 771). Sequencing results showed that three novel single nucleotide polymorphisms (SNPs) were identified at position 44087 (G/A), 65550 (G/A), and 65676(G/A) in the PPARα gene. PCR–restriction fragment length polymorphism technology was used to genotype the three SNPs. Association analysis showed that cows with H1H8 (P < 0.05), H2H8 (P < 0.01), H5H7 (P < 0.05), H5H8 (P < 0.05), and H8H8 (P < 0.05) haplotype combinations had lower potassium content in erythrocytes than those with H2H6 haplotype combination. Cows with H1H8, and H8H8 haplotype combinations had lower decrease rate of milk yield than those with H2H6 and H6H8 haplotype combinations (P < 0.05). Cows with H2H8 and H8H8 haplotype combinations had lower rectal temperature than those with H5H8 and H7H7 haplotype combinations (P < 0.05). In conclusion, H8H8 haplotype combination may be advantageous for heat resistance traits in Chinese Holstein cattle.     

Polymorphism of β-lacto globulin gene in Indian goats and its effect on milk yield

Polymorphism in the beta-LG gene in the Indian goat was investigated by the SDS-PAGE and PCR-RFLP method. SDS-PAGE was carried out in 1098 samples belonging to 8 different breeds of Indian goats. The electrophoretic pattern in the beta-LG locus showed the presence of AA and AB genotypes with frequencies of 0.81 and 0.19, 0.89 and 0.11, 0.50 and 0.50, 0.80 and 0.20, 0.84 and 0.16, 1.00 and 0.00, 0.98 and 0.02 and 0.950 and 0.050 in Jamunapari, Barbari, Marwari, Sirohi, Jakhrana, Beetal, Local UP and Local MP goats, respectively. A total of 358 individuals belonging to 13 different genetic groups were analyzed by the PCR-RFLP method. The amplified product was observed as 426 bp and the restriction digestion with SacII revealed three genotypes, namely S1S1, S1S2 and S2S2 at the beta-LG locus. The frequency of the S2S2 genotype ranged from 0.42 to 1.00 in the population. An analysis was carried out in Jamunapari and Barbari goats to observe the effect of the beta-LG genotype on 90-day milk yield. Least squares analysis of data showed that beta-LG AA animals had higher milk yield than the beta-LG AB genotype in both breeds (P < 0.01).     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.