Selective growth of malignant cells by in vitro incubation on Teflon

The selective growth of malignant cells on non-adhesive Teflon substrates is reported. Neoplastically transformed cell lines of human and murine origin proliferated on Teflon whereas similarly seeded normal non-tumorigenic cells were unable to undergo cell division and subsequent growth. This substrate should be especially useful in establishing primary cultures from human tumor material.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

Gonadectomy in Mito-Ob mice revealed a sex-dimorphic relationship between prohibitin and sex steroids in adipose tissue biology and glucose homeostasis

Background

Recently, we have developed a novel transgenic mouse model by overexpressing prohibitin (PHB) in adipocytes, which developed obesity due to upregulation of mitochondrial biogenesis in adipocytes, hence named “Mito-Ob.” Interestingly, only male Mito-Ob mice developed obesity-related impaired glucose homeostasis and insulin sensitivity, whereas female Mito-Ob mice did not. The observed sex differences in metabolic dysregulation suggest a potential involvement of sex steroids. Thus, the main aim of this study is to investigate the role of sex steroids on the overall phenotype of Mito-Ob mice through gonadectomy, as well as direct effect of sex steroids on adipocytes from Mito-Ob mice in vitro.

Methods

Mito-Ob mice and wild-type CD-1 mice were gonadectomized at 12 weeks of age. Age- and sex-matched sham-operated mice were used as controls. Body weight, white adipose tissue, glucose tolerance, and insulin sensitivity were analyzed 3 months post-surgery. Differentiation of adipocytes isolated from female and male Mito-Ob mice were studied with and without sex steroids.

Results

Gonadectomy significantly reduced body weight in Mito-Ob mice compared with sham-operated mice, whereas the opposite trend was observed in wild-type mice. These changes occurred independent of food intake. A corresponding decrease in adipose tissue weight was found in gonadectomized Mito-Ob mice, but depot-specific differences were observed in male and female. Gonadectomy improved glucose tolerance in male wild-type and Mito-Ob mice, but the effect was more pronounced in wild-type mice. Gonadectomy did not alter insulin sensitivity in male Mito-Ob mice, but it was improved in male wild-type mice. In primary cell cultures, testosterone inhibited adipocyte differentiation to a lesser extent in male Mito-Ob preadipocytes compared with the wild-type mice. On the other hand, preadipocytes from female wild-type mice showed better differentiation potential than those from female Mito-Ob mice in the presence of 17β-estradiol.

Conclusions

PHB requires sex steroids for the development of obese phenotype in Mito-Ob mice, which differentially affect glucose homeostasis and insulin sensitivity in male and female. It appears that PHB plays sex- and adipose depot-specific roles and involves additional factors. In vitro studies suggested that PHB differently influenced adipocyte differentiation in the presence and absence of sex steroids. Overall, this study along with available information in the literature indicated that a multifaceted relationship exists between PHB and sex steroids, which may work in a cell/tissue type- and sex-specific manner.

     

Investigation of Trace Element Content in the Seeds,Pulp, and Peel of Mashui Oranges Using Microwave Digestion and ICP-MS Analysis

Fresh Mashui orange samples were pretreated with microwave digestion using an HNO₃-H₂O₂ system. The levels of Mg, K, Ca, Fe, Mn, Cu, Zn, As, Cd, and Pb in the seeds, pulp, and peel were then determined using inductively coupled plasma mass spectrometry (ICP-MS) combined with collision cell technology (CCT) and kinetic energy discrimination (KED). The standard curve coefficient of determinations of the ten tested elements were between 0.9995 and 0.9999. The instrument detection limit was between 0.112 ng/L and 3.05 ng/mL. The method detection limit was between 0.0281 and 763 ng/g. The average recovery rate was between 85.0 and 117%. The current results showed that Mashui oranges are rich in three elements, namely Mg, K, and Ca. The concentrations of K and Ca were significantly higher than that of Mg in the peel. The content of K was the highest in the seeds. Fe, Mn, Cu, and Zn had the second highest concentrations, and Fe was the highest in the seeds, while Cu was the lowest in the peel. As, Cd, and Pb (hazardous elements) had the lowest concentrations of all the tested elements.     

p21 Inhibits Cdk1 in the absence of Cdk2 to maintain the G1/S phase DNA damage checkpoint

Cdk1 was proposed to compensate for the loss of Cdk2. Here we present evidence that this is possible due to premature translocation of Cdk1 from the cytoplasm to the nucleus in the absence of Cdk2. We also investigated the consequence of loss of Cdk2 on the maintenance of the G1/S DNA damage checkpoint. Cdk2(-/-) mouse embryonic fibroblasts in vitro as well as regenerating liver cells after partial hepatectomy (PH) in Cdk2(-/-) mice, arrest promptly at the G1/S checkpoint in response to gamma-irradiation due to activation of p53 and p21 inhibiting Cdk1. Furthermore re-entry into S phase after irradiation was delayed in Cdk2(-/-) cells due to prolonged and impaired DNA repair activity. In addition, Cdk2(-/-) mice were more sensitive to lethal irradiation compared to wild-type and displayed delayed resumption of DNA replication in regenerating liver cells. Our results suggest that the G1/S DNA damage checkpoint is intact in the absence of Cdk2, but Cdk2 is important for proper repair of the damaged DNA.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Pollution characteristics and health risk assessment of potentially toxic elements in school playground soils: A case study of Lagos,Nigeria

Abstract

Soil samples were collected in February 2014 from 25 school playgrounds in Lagos, Nigeria to assess the potential adverse effects of the exposure of children to potentially toxic element (PTE). In each of the playgrounds, about 500?g of bulked soil samples were collected, dried, sieved, acid digested, and analyzed by inductively coupled plasma mass spectrometry (ICP‐MS). Results showed that soils studied were characteristically unpolluted as the average PTE concentration at each site did not exceed the soil guideline values. Considering the pollution assessment tools employed, some soil samples showed some form of anthropogenic input from PTE. Health risk assessment was employed to assess PTE exposure from ingestion, inhalation, and dermal contact. Result indicated that the highest risk is associated with ingestion followed by dermal contact and inhalation. For non‐carcinogenic effects, exposure to school playground soils poses no threat to children as the overall value of hazard index is less than the safe level of 1. For carcinogenic effects, only Cr and Ni were considered and were below the threshold of 1?×?10^–6. This study has demonstrated that minimal risk arises from the investigated playgrounds and that regular monitoring is required to keep the PTE contents low in soils to avoid risk to human health.

Abbreviations: SH, school playground; PTE, potentially toxic elements