Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides

The acid ceramidase (AC) inhibitory activity of octanoylamides, p-tert-butylbenzamides and pivaloylamides of several 2-substituted aminoethanols is reported. All the aminoethanol amides bearing a hexadecyl substituent (C16), as well as (S)-N-(1-(hexadecylthio)-3-hydroxypropan-2-yl)pivaloylamide (SC16-tb) were inhibitory in cell lysates overexpressing AC, while all other compounds were not inhibitors. Kinetic experiments with (R,E)-N-(1-hydroxyoctadec-3-en-2-yl)pivaloylamide (E-tb) and SC16-tb showed that inhibition was competitive, with K(i) values of 34 and 94.0 microM, respectively. None of the compounds inhibited neutral ceramidase. Compounds E-tb and E-c7 (the octanoylamide of the unsaturated base E), which elicited a dose-response inhibition with IC(50) values around 15 microM, were the only AC inhibitors in intact cells. Both compounds were toxic to A549 cells with LD(50) values nearly 40 microM. Flow cytometry studies with E-tb evidenced that this compound induced a concentration-dependent cell cycle arrest at G(1) and a 20-25% apoptosis/late apoptosis/necrosis after a 24-h incubation at 50 microM. In agreement with its activity as acidic ceramidase inhibitor, this effect was accompanied with an increase in the amounts of C14, C16 and C18 ceramides (LC-MS analyses), which suggested that these lipids may be responsible for the cytotoxic activity of E-tb.     

Optimization of a fermentation process for bioinsecticide production by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

In an attempt to develop a cost-effective process for bioinsecticide production by B. thuringiensis, the feeding regime during aerobic cultivation of the bacterium was investigated and optimized. The process was designed as a two-stage process; a first stage of active growth, where glucose and other nutrients were adequately supplied to the growing cells over 12 h, followed by a second stage of 2 h for spore formation and toxin release. In order to maximize spore and toxin yield and productivity, different quantities of glucose and nutrients were fed separately to the growing cells in four different fermentation runs. In all runs, glucose was converted to bacterial biomass during the first stage and subsequently to spores and crystal protein during the second phase. The best results were obtained with a fermentation run supplied with 190 g glucose in 1500 ml. Up to 20.1 g of bacterial insecticides/l were recovered from fermentation broth with a glucose to toxin conversion yield of 0.159 g/g. Also, a markedly high spore concentration of 2.31 × 10¹² c.f.u./ml was obtained. The spore–crystal protein mixture obtained was tested for its insecticidal activity against three of the most agronomically important pests. Among the bioinsecticide-treated insect pests, Egyptian cotton leafworm, Spodoptera littoralis was the most susceptible pest with the lowest LC₅₀ of the bioinsecticides against its larval instar and the highest virulence against adults emerged later on from the surviving larvae.     

Evolutionarily conserved thermal biology across continents: The North American lizard Plestiodon gilberti (Scincidae) compared to Asian Plestiodon

1.

The lizard genus Plestiodon (Scincidae; formerly Eumeces) is widespread in North America and Asia, but the thermal biology of only a few species has been studied.     

Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan

It was observed that levansucrase from Bacillus natto became unstable and was easily inactivated when the salts were removed from the enzyme solution, while the enzyme was stable for long time in a buffered saline. After modification with periodate oxidized yeast glucomannan, the enzyme increased thermal stability up to 45°C, in which it conserved more than 90% of its activity after 15 min treatement. The optimum temperature was also shifted from 40°C in the case of original enzyme to 50°C for the modified enzyme after 10 min reaction time. The half-life time increased significantly from 9 min to 55 min at 50°C, however it increased from 30 min and 22 min respectively at 40°C and 45°C to more than 1 h at the same temperature. The content of carbohydrates of modified enzyme was 25% that increases the molecular weight from 57 KDa to 80 KDa. The products from sucrose by the modified enzyme were the same as the case using original enzyme. Namely, the products confirmed were levan and 3 kestoses (6-, 1-, and neo-kestose).     

Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.     

Targeting PI3K/p-Akt/eNOS,Nrf2/HO-1, and NF-κB/p53 signaling pathways by angiotensin 1–7 protects against liver injury induced by ischemia–reperfusion in rats

The liver is an important organ, and hepatic ischemia–reperfusion (IR) injury is a frequent pathophysiological process that can cause significant morbidity and mortality. Thus, our study aimed to investigate the effect of targeting PI3K/p-Akt/eNOS (phosphoinositide 3-kinase/phospho-protein kinase B/endothelial nitric oxide synthase), Nrf2/HO-1 (nuclear factor-erythroid 2-related factor-2/heme oxygenase-1), and NF-κB/p53 (nuclear factor-κB/tumor protein 53) signaling pathways by using angiotensin (1–7) [ang-(1–7)] against hepatic injury induced by IR. Thirty-two male rats were included in sham group, ang-(1–7)-treated group, hepatic IR group, and hepatic IR group treated with ang-(1–7). The levels of hepatic ang-(1–7), angiotensin II (Ang II), angiotensin-converting enzyme 2 (ACE2), HO-1, malondialdehyde (MDA), PI3K, and p-Akt were assessed. The expressions of eNOS and B-cell leukemia/lymphoma-2 (BCL-2) in the liver were determined. Histological assessment and immunohistochemical expression of NF-κB, p53, and Nrf2 were carried out. The levels of reduced glutathione (GSH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in serum were estimated. Results showed that administration of ang-(1–7) to hepatic IR rats led to significant amelioration of hepatic damage through a histological evaluation that was associated with significant upregulation of the expressions of PI3K/p-Akt/eNOS and Nrf2/HO-1 with downregulation of NF-κB/p53 signaling pathways. In conclusion, PI3K/p-Akt/eNOS and Nrf2/HO-1 signaling pathways are involved in the protective effects of ang-(1–7) against hepatic damage induced by IR. Therefore, ang-(1–7) can be used to prevent hepatic IR, which occurs in certain conditions such as liver transplantation, hemorrhagic shock, and severe infection.     

The miRNA-kallikrein axis of interaction: a new dimension in the pathogenesis of prostate cancer

Kallikrein-related peptidases (KLKs) are a family of serine proteases that were shown to be useful cancer biomarkers. KLKs have been shown to be dysregulated in prostate cancer (PCa). microRNAs (miRNAs) are short RNA nucleotides that negatively regulate gene expression and have been reportedly dysregulated in PCa. We compiled a comprehensive list of 55 miRNAs that are differentially expressed in PCa from previous microarray analysis and published literature. Target prediction analyses showed that 29 of these miRNAs are predicted to target 10 KLKs. Eight of these miRNAs were predicted to target more than one KLK. Quantitative real-time (qRT)-PCR demonstrated that there was an inverse correlation pattern in the expression (normal vs. cancer) between dysregulated miRNAs and their target KLKs. In addition, we experientially validated the miRNA-KLK interaction by transfecting miR-331-3p and miR-143 into a PCa cell line. Decreased expression of targets KLK4 and KLK10, respectively, and decreased cellular growth were observed. In addition to KLKs, dysregulated miRNAs were predicted to target other genes involved in the pathogenesis of PCa. These data show that miRNAs can contribute to KLK regulation in PCa. The miRNA-KLK axis of interaction projects a new element in the pathogenesis of PCa that may have therapeutic implications.