Plasma membrane homing of tissue nonspecific alkaline phosphatase under the influence of 3-hydrogenkwadaphnin, an antiproliferative agent from Dendrostellera lessertii

Several mammalian enzymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) structure. These include acetylcholinesterase, alkaline phosphatase (AP) and 5'-nucleotidase among other enzymes. Recently, it has been reported that these membrane enzymes can be released into the serum by the GPI-dependent phospholipase D under various medical disturbances such as cancer and/or by chemical and physical manipulation of the biological systems. Treatment of MCF-7 cells with two consecutive effective concentrations of 3-hydrogenkwadaphnin (3-HK, 3 nM) for 48 h enhanced membrane AP activity by almost 330% along with a 40% reduction in the AP activity of the cell culture medium. In addition, our data indicate that 3-HK is capable of inducing mainly the tissue-nonspecific alkaline phosphatase (TNAP) isoenzyme, along with enhancing its thermostability. These findings, besides establishing a correlation between the antiproliferative activity of 3-HK and the extent of plasma membrane AP activity, might assist in the development of new diagnostic tools for following cancer medical treatments.     

Optimization of conformational stability and catalytic efficiency in chondroitinase ABC Ι by protein engineering methods

Chondroitinase ABC Ι can promote the recovery of spinal cord injuries by depolimerization of glycosaminoglycans. However, low thermal stability is one of the limitations regarding its clinical application. In order to increase the conformational stability of the enzyme, Leu⁶⁷⁹ at the starting point of a short helix located at the C‐terminal domain of the protein was replaced by serine (L679S mutant) and aspartic acid (L679D mutant). Theoretical and spectroscopic studies showed that the stability of enzyme increased upon mutation. Based on the activity measurements, the catalytic efficiency of L679S was improved in comparison with the wild‐type protein; while that of L679D (a more stabilized protein) was not changed. According to the structural and kinetic data, we proposed a model in which a higher conformational stability results in a slower rate of the formation of the open conformation. On the other hand, a higher flexibility slows down the rate of the formation and holding of the closed conformation. Therefore, the L679S mutant, which is structurally stable relative to the wild‐type protein and is destabilized compared to the L679D mutant, exhibited the best catalytic efficiency. However, it was also found that the L679D mutant was more suitable for long‐term storage of the enzyme.     

Analysis of RAS gene mutations in cytogenetically normal de novo acute myeloid leukemia patients reveals some novel alterations

Rat sarcoma gene (RAS) holds great importance in pathogenesis of acute myeloid leukemia (AML). The activated mutations in Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) and Kirsten rat sarcoma viral oncogene homolog (KRAS) confers proliferative and survival signals, deliberating numerous effects on overall survival and progression free survival in AML patients. In this study thirty one (31) blood samples of adult newly diagnosed AML patients were collected to identify possible incidence of mutations through amplification of KRAS (exon 1 and 2) and NRAS gene (exon 1 and 2) using polymerase chain reaction (PCR). Amplicons were then subjected to sequencing and were analyzed through Geneious Prime 2019. Five of thirty one (16.12%) patients had altered sites in either NRAS or KRAS. The NRAS mutations were observed in three AML patients (N = 3, 9.67%). A novel missense mutation NRAS-I36R (239 T > G) representing a substitution of single nucleotide basepair found in NRAS exon 1 while exon 2 was detected with heterozygous mutation NRAS-E63X (318G > T) and insertion (A), resulting in frameshift of the amino acid sequence and insertion of two nucleotide basepairs (TA) in two of the patients. KRAS mutations (N = 2, 6.45%) were found in exon 1 whereas no mutations in KRAS exon 2 were detected in our patient cohort. Mutation in KRAS Exon 1, KRAS-D30N (280G > A) was observed in two patients and one of them also had a novel heterozygous mutation KRAS-L16N (240G > C). In addition there was no statistically significant association of mutRAS gene of AML patients with several prognostic markers including age, gender, karyotyping, CD34 positivity, cytogenetic abnormalities, total leukocyte count, white blood cell count and French-American-British (FAB) classification. However, the presence of mutRAS gene were strongly associated (p = 0.001) with increased percentage of bone marrow blasts. The prevalence of mutations in correlation with clinical and hematological parameter is useful for risk stratification in AML patients.     

The Effect of Temperature on the Toxicity of Insecticides against Musca domestica L.: Implications for the Effective Management of Diarrhea

Background

Diarrhea is an important cause of childhood mortality in developing countries like Pakistan because of unhygienic conditions, lack of awareness, and unwise use of preventive measures. Mechanical transmission of diarrheal pathogens by house flies, Musca domestica, is believed as the most effective route of diarrhea transmission. Although the use of insecticides as a preventive measure is common worldwide for the management of house flies, success of the measure could be compromised by the prevailing environmental temperature since it significantly affects toxicity of insecticides and thus their efficacy. Peaks of the house fly density and diarrheal cases are usually coincided and season specific, yet little is known about the season specific use of insecticides.

Methodology/Principal Findings

To determine the temperature-toxicity relationship in house flies, the effect of post-bioassays temperature (range, 20–34°C) on the toxicity of seven insecticides from organophosphate (chlorpyrifos, profenofos), pyrethroid (cypermethrin, deltamethrin) and new chemical (emamectin benzoate, fipronil, spinosad) classes was evaluated by using a feeding bioassay method. From 20–34°C, the toxicities of chlorpyrifos, profenofos, emamectin and fipronil increased 2.10, 2.93, 2.40 and 3.82 fold (i.e. positive temperature coefficient), respectively. Whereas, the toxicities of cypermethrin, deltamethrin and spinosad decreased 2.21, 2.42 and 3.16 fold (i.e. negative temperature coefficient), respectively.

Conclusion/Significance

These findings suggest that for the reduction in diarrheal cases, house flies should be controlled with insecticides according to the prevailing environmental temperature. Insecticides with a positive temperature coefficient may serve as potential candidates in controlling house flies and diarrhea epidemics in hot season and vice versa.     

Dihydro-CDDO-trifluoroethyl amide suppresses inflammatory responses in macrophages via activation of Nrf2

Nuclear factor erythroid 2-related factor (Nrf2) is the major regulator of cellular defenses against various pathological stresses in a variety of organ systems, thus Nrf2 has evolved to be an attractive drug target for the treatment and/or prevention of human disease. Several synthetic oleanolic triterpenoids including dihydro-CDDO-trifluoroethyl amide (dh404) appear to be potent activators of Nrf2 and exhibit chemopreventive promises in multiple disease models. While the pharmacological efficacy of Nrf2 activators may be dependent on the nature of Nrf2 activation in specific cell types of target organs, the precise role of Nrf2 in mediating biological effects of Nrf2 activating compounds in various cell types remains to be further explored. Herein we report a unique and Nrf2-dependent anti-inflammatory profile of dh404 in inflamed macrophages. In lipopolysaccharide (LPS)-inflamed RAW264.7 macrophages, dh404 dramatically suppressed the expression of pro-inflammatory cytokines including inducible nitric oxide synthase (iNOS), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β), while minimally regulating the expression of interleulin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNFα). Dh404 potently activated Nrf2 signaling; however, it did not affect LPS-induced NF-κB activity. Dh404 did not interrupt the interaction of Nrf2 with its endogenous inhibitor Kelch-like ECH associating protein 1 (Keap1) in macrophages. Moreover, knockout of Nrf2 blocked the dh404-induced anti-inflammatory responses in LPS-inflamed macrophages. These results demonstrated that dh404 suppresses pro-inflammatory responses in macrophages via an activation of Nrf2 independently of Keap1 and NF-κB, suggesting a unique therapeutic potential of dh404 for specific targeting a Nrf2-mediated resolution of inflammation.     

The biological effects of vanadyl curcumin and vanadyl diacetylcurcumin complexes: the effect on structure,function and oxidative stability of the peroxidase enzyme,antibacterial activity and cytotoxic effect

Curcumin has multiple pharmacological effects, but it has poor stability. Complexation of curcumin with metals improves its stability. Here, the effects of vanadyl curcumin and vanadyl diacetylcurcumin on the function and structure of horseradish peroxidase enzyme were evaluated by spectroscopic techniques. Cytotoxic effect of the complexes was also assessed on MCF-7 breast cancer, bladder and LNCaP prostate carcinoma cell line. The results showed that the complexes improve catalytic activity of HRP, and also increase its tolerance against the oxidative condition. The result also indicated that the affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism and fluorescence spectroscopies showed that compactness of the enzyme structure around the catalytic heme group and the distance between the heme group and tryptophan residue decreases after the binding. The antibacterial and cytotoxic results indicated that the complexes have anticancer potential, but they have no considerable antibacterial activity.     

Altered serum pro-inflammatory cytokines in children with Down's syndrome

There are reports showing that pro-inflammatory cytokines are dysregulated in patients with Down's syndrome (DS). However, most of these reports concern adults. We analyzed cytokine levels in serum samples from children with DS, and compared them with samples from intellectually disabled (ID), and healthy, control children. Blood samples were collected from 24 DS, 24 age-/sex-matched ID, and 24 age-/sex-matched healthy, control children. Serum levels of the cytokines IL-5, IL-10, IL-13, IFN-γ, and TNF-α were measured using a sandwich ELISA method, . The age range of the children was 1-15 years, with a mean ± SD of 5.75 ± 4.36 years. TNF-α levels were significantly higher in the DS and ID groups compared with those found in healthy, control children (P<0.05). The DS and ID groups had significantly higher IFN-γ levels compared with healthy, control children (P = 0.0002 and P<0.01, respectively), with significant higher levels in the DS than the ID group (P<0.05). Serum from the ID group showed significantly higher IL-10 levels compared with those from the DS group (P<0.05), but not the healthy, control group. Significant correlations were found between the differences in TNF-α and IFN-γ levels, in both ID (rs = 0.558; P = 0.005) and DS children (rs = 0.405; P<0.05). There were no significant differences found in serum levels of IL-13 between the groups, and IL-5 was not detectable in any of the serum samples. Levels of TNF-α and IFN-γ were increased, and IL-10 decreased in serum from children with DS. It may be that these differences contribute to the clinical symptoms seen in DS: consequently, these pro-inflammatory cytokines might be useful as early biomarkers of the disorders associated with DS.     

Alpha-Tocopherol-Induced Regulation of Growth and Metabolism in Plants Under Non-stress and Stress Conditions

Alpha-tocopherol (α-Toc) is a member of the vitamin E family and is lipid soluble. Its biosynthesis is by the reaction of isopentyl diphosphate and homogentisic acid in plastid membranes. The putative biochemical activities of tocopherols are linked with the formation of tocopherol quinone species, which subsequently undergo degradation and recycling within cells/tissues. α-Toc plays a key role in a variety of plant metabolic processes throughout the ontogeny of plants. It can maintain the integrity and fluidity of photosynthesizing membranes. It can also neutralize lipid peroxy radicals, consequently blocking lipid peroxidation by quenching oxidative cations. It preserves membrane integrity by retaining membranous structural components under environmental constraints such as water deficiency, high salt content, toxic metals, high/low temperatures, and radiations. α-Toc also induces cellular signalling pathways within biological membranes. Its biosynthesis varies during growth and developmental stages as well as under different environmental conditions. The current review primarily focuses on how α-Toc can regulate various metabolic processes involved in promoting plant growth and development under stress and non-stress and how it can effectively counteract the stress-induced high accumulation of reactive oxygen species (ROS). Currently, exogenous application of α-Toc has been widely reported as a potential means of promoting resistance in plants to a variety of stressful environments.