NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca(2+) oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.     

Analysis of extracellular proteins of two Perkinsus spp. isolated from the softshell clam Mya arenaria in vitro

Biochemical characterization of the extracellular proteins (ECP) of two softshell clam Perkinsus spp. cloned isolates, Perkinsus chesapeaki isolate G-117 and Perkinsus marinus H-49, was performed and compared to that of the oyster-derived P. marinus isolate P-1. G-117 and H-49 demonstrated distinct differences in enzyme activities; however, all three isolates shared common bands. Substrate-impregnated gels showed H-49 to possess proteolytic activities while G-117 did not. Inhibition studies revealed that H-49 ECP contain serine proteases similar to those described for P-1. The G-117 ECP lacked proteolytic activity but showed a higher production of lipolytic enzymes than H-49 or P-1. Optimal in vitro growth temperatures for the two clam isolates were generally lower than those for P-1. G-117 showed faster growth at lower salinities than either H-49 or P-1. Clam Perkinsus spp. isolates appear to be better adapted to lower salinities and temperatures than the P. marinus isolate of the eastern oyster.     

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.     

Improved Movat Pentachrome Stain

Movat in 1955 developed a staining method which demonstrates collagen fibers, mucin, muscle fibers, elastic fibers, fibrin and fibrinoid changes in a single section. His procedure was considered excellent by Lynch et al. (1969)     

Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection

Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.     

Cytokines as potential combination agents with PD-1/PD-L1 blockade for cancer treatment

Immunotherapy has caused a paradigm shift in the treatment of several malignancies, particularly the blockade of programmed death-1 (PD-1) and its specific receptor/ligand PD-L1 that have revolutionized the treatment of a variety of malignancies, but significant durable responses only occur in a small percentage of patients, and other patients failed to respond to the treatment. Even those who initially respond can ultimately relapse despite maintenance treatment, there is considerable potential for synergistic combinations of immunotherapy and chemotherapy agents with immune checkpoint inhibitors into conventional cancer treatments. The clinical experience in the use of cytokines in the clinical setting indicated the efficiency of cytokine therapy in cancer immunotherapy. Combinational approaches to enhancing PD-L1/PD-1 pathways blockade efficacy with several cytokines such as interleukin (IL)-2, IL-15, IL-21, IL-12, IL-10, and interferon-α (IFN-α) may result in additional benefits. In this review, the current state of knowledge about PD-1/PD-L1 inhibitors, the date in the literature to ascertain the combination of anti-PD-1/PD-L1 antibodies with cytokines is discussed. Finally, it is noteworthy that novel therapeutic approaches based on the efficient combination of recombinant cytokines with the PD-L1/PD-1 blockade therapy can enhance antitumor immune responses against various malignancies.