Role of ataxia telangiectasia mutated in insulin signalling of muscle-derived cell lines and mouse soleus.

AIM: Ataxia telangiectasia mutated (ATM) reportedly plays a role in insulin-stimulated activation of Akt in some cell types but not in others. The role of ATM in insulin signalling has not been firmly resolved for skeletal muscle cells, for which Akt phosphorylation is a pivotal step in stimulation of glucose transport. Accordingly, our aim was to determine the role of ATM in insulin effects for cell lines derived from skeletal muscle and for skeletal muscle. METHODS: We examined insulin effects in L6 myotubes, mouse soleus, C2C12 myotubes and differentiated rhabdomyosarcoma (RD) cells in the presence and absence of a low concentration (1 microm) of the ATM inhibitor KU55933. We also compared insulin signalling in C2C12 cells expressing shRNA against ATM and control cell lines (empty vector; cells expressing non-targeting shRNA). RESULTS: In L6 myotubes and mouse soleus muscle, KU55933 inhibited insulin-stimulated phosphorylation of the 160 kDa substrate of Akt (AS160) despite no effect on Akt. In contrast, KU55933 prevented insulin-stimulated Akt phosphorylation in C2C12 myotubes. Furthermore, C2C12 myotubes expressing shRNA against ATM displayed reduced insulin-stimulated Akt phosphorylation compared to controls. KU55933 also decreased insulin-stimulated Akt phosphorylation in differentiated RD cells. CONCLUSION: These model-dependent differences in the role of ATM in insulin action demonstrate a role of ATM in insulin-stimulated phosphorylation of Akt (in C2C12 and RD cells) but also allow the elucidation of a novel, Akt-independent role of ATM (in L6 myotubes and mouse soleus, at the level of AS160) in insulin signalling.

Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.

Differential induction of sesquiterpene metabolism in tobacco cell suspension cultures by methyl jasmonate and fungal elicitor.

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Technical basis for EPA's proposed regulation on the cleanup of sites contaminated with radioactivity.

The U.S. Environmental Protection Agency is proposing a regulation for the protection of the public from radioactive contamination at sites that are to be cleaned up and released for public use. The rule will apply to sites under the control of Federal agencies, and will impose limits on radiation doses to individuals living or working on a site following cleanup; it will thereby provide site owners and managers with uniform, consistent cleanup criteria for planning and carrying out remediation. This paper presents an overview of EPA's approach to assessing some of the beneficial and adverse effects associated with various possible values for the annual dose limit. In particular, it discusses the method developed to determine how the choice of cleanup criterion affects (1) the time-integrated potential numbers of non-fatal and fatal radiogenic cancers averted among future populations, (2) the occurrence of radiogenic cancers among remediation workers and the public caused by the cleanup process itself, and (3) the volumes of contaminated soil that may require remediation. The analytic methods described here were used to provide input data and assumptions for the Regulatory Impact Analysis (RIA) that supports the proposed regulation; the RIA also considered non-radiological benefits and costs (i.e., public health, economic, and ecological) of the standards.

L-trans-2,3-pyrrolidine dicarboxylate: characterization of a novel excitotoxin.

This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-glutamate, L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 h) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 microM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 microM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 microM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transported substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes.

Polonium metabolism in adult female baboons.

The biokinetics of polonium in nonhuman primates (Papio anubis) has been studied after intravenous injection of 210Po citrate. The urinary excretion of polonium in the baboon could be described by a single exponential function with a half-time of 15.6 days. Excretion fractions of polonium were found to be markedly different from those reported for other species, including humans. Polonium-210 was found to be distributed throughout the soft tissues of the baboon with 29% of the injected polonium being deposited in liver, 7% in kidneys and 0.6% in spleen. Retention of polonium in all tissues exhibited single exponential functions; however, the biological half-times were variable, ranging from 15 to 50 days.

The importance of acid digestion of urine prior to spontaneous deposition of 210Po.

Historically, radiochemical analysis of 210Po in urine has used spontaneous deposition of the nuclide directly from raw urine onto a suitable metal disc. Consequently, the urinary excretion fraction for Po in some current metabolic and dosimetric models is based on studies which inherently assume that metabolized (i.e., filtered out of the blood by the kidneys) 210Po is plated with the same efficiency as tracer 210Po which has been added to urine samples. Urine samples collected after intravenous administration of 210Po citrate to two species of nonhuman primates were divided and simultaneously analyzed via two methods: the historical procedure of plating 210Po from raw urine for one sample and a method which includes the addition of 208Po tracer and sample digestion with concentrated HNO3 prior to 210Po deposition for the other sample. A more significant amount of 210Po was consistently recovered when the urine was wet ashed then when it was not wet ashed. A temporal relationship was found to describe the change in the ratio of the deposition recoveries for the two methods. Possible mechanisms for this phenomenon and its dosimetric implications are discussed.

Transparent electrophotographic films for optical data storage applications.

The experimental evaluation of a transparent electrophotographic (TEP) film is reported. The objective of the evaluation was to determine the suitability of TEP films for optical data storage applications, especially those using holography. The characteristics of TEP films that motivated the evaluation are good sensitometric and holographic recording responses, relatively high resolution, and a number of attractive applications properties, e.g., long shelf life, good environmental integrity, and exceptional image permanence. In addition, TEP films have an add-on capability. A general description of TEP films is given, and sensitometric and holographic data are summarized for an experimental TEP film.