Comparative effects of interferon alpha‐2b and pegylated interferon alpha‐2b on menstrual cycles and ovarian hormones in cynomolgus monkeys

BACKGROUND : The covalent modification of interferon (IFN) α2b with monomethyoxy polyethylene glycol (PEG) reduces its clearance rate and increases its half‐life. High doses of interferon (IFN) α2b have previously been shown to affect maintenance of pregnancy in rhesus monkeys. Given the role of ovarian hormones in reproductive function and pregnancy, this study was conducted to assess the effects of PEG‐IFNα2b or IFNα2b (comparative control) on ovarian hormones and menstrual cyclicity in cynomolgus monkeys. In addition, the potential for reversibility of PEG‐IFNα2b or IFNα2b‐related observations was assessed. METHODS : Monkeys were administered 3,105 µg/m² human recombinant (hr) IFNα2b or 52, 262, or 4,239 µg/m² PEG‐hr‐IFNα2b every other day for one menstrual cycle, followed by a post‐dose period of up to two menstrual cycles. RESULTS : Monkeys administered 3,105 µg/m² hr‐IFNα2b or 52, 262, or 4,239 µg/m² PEG‐hr‐IFNα2b exhibited transient decreases in food consumption, leukocyte and erythrocyte parameters. Monkeys administered 3,105 µg/m² hr‐IFNα2b exhibited lengthened menstrual cycles that were associated with a delay in reaching peak ovarian hormone levels and lower respective peak concentrations. Similarly, monkeys administered 4,239 µg/m² PEG‐hr‐IFNα2b exhibited lengthened menstrual cycles and a delay in reaching peak ovarian hormone levels and slightly lower respective peak concentrations. Post‐dosing menstrual cycle length, estradiol and progesterone profiles exhibited evidence of recovery in both the hr‐IFNα2b and the high‐dose PEG‐hr‐IFNα2b groups. CONCLUSIONS : Administration of hr‐IFNα2b or PEG‐hr‐IFNα2b at high doses to cynomolgus monkeys resulted in similar effects on menstrual cycles, estradiol and progesterone profiles, and exhibited evidence of reversibility upon cessation of dosing. These results suggest that the previously observed high‐dose IFNα‐related effects on the maintenance of pregnancy in monkeys are likely the result of altered ovarian function. Birth Defects Res (Part B) 86:29‐39, 2009. © 2009 Wiley‐Liss, Inc.     

Eucalyptus recruitment in degraded woodlands: no benefit from elevated soil fertility

Grasses and forbs compete heavily with young tree seedlings for available resources, greatly reducing tree seedling establishment success. Soil nutrient enrichment associated with agricultural intensification can increase the growth of both herbaceous and woody lifeforms growing in isolation, but may change the balance of competitive advantage when growing together. The effects of nitrogen and phosphorus enrichment on pasture biomass and competition with two Australian grassy woodland trees (Eucalyptus albens and Eucalyptus microcarpa) was investigated in a field plot trial. Soil nutrients increased pasture biomass, but had no measurable effect on tree growth in our experiment. Competition from pasture species, even at low levels, led to high tree seedling mortality and greatly reduced tree seedling growth compared with pasture-free plots. However, when pasture-free plots were excluded from the analysis, tree seedling leaf area was not strongly correlated with herbaceous biomass. Tree seedling establishment was severely restricted even at the lowest levels of pasture biomass. We conclude that increased soil fertility resulted in a competitive advantage to the pasture, and does not improve tree seedling establishment when grown either with or without exotic herbaceous pasture (grassy understorey) species.     

Chronic Alcohol Exposure Increases Circulating Bioactive Oxidized Phospholipids

Ethanol metabolism by liver generates short lived reactive oxygen species that damage liver but also affects distal organs through unknown mechanisms. We hypothesized that dissemination of liver oxidative stress proceeds through release of biologically active oxidized lipids to the circulation. We searched for these by tandem mass spectrometry in plasma of rats fed a Lieber-DeCarli ethanol diet or in patients with established alcoholic liver inflammation, steatohepatitis. We found a severalfold increase in plasma peroxidized phosphatidylcholines, inflammatory and pro-apoptotic oxidatively truncated phospholipids, and platelet-activating factor, a remarkably potent and pleiotropic inflammatory mediator, in rats chronically ingesting ethanol. Circulating peroxidized phospholipids also increased in humans with established steatohepatitis. However, reactive oxygen species generated by liver ethanol catabolism were not directly responsible for circulating oxidized phospholipids because the delayed appearance of these lipids did not correlate with ethanol exposure, hepatic oxidative insult, nor plasma alanine transaminase marking hepatocyte damage. Rather, circulating oxidized lipids correlated with steatohepatitis and tumor necrosis factor-α deposition in liver. The organic osmolyte 2-aminoethylsulfonic acid (taurine), which reduces liver endoplasmic reticulum stress and inflammation, even though it is not an antioxidant, abolished liver damage and the increase in circulating oxidized phospholipids. Thus, circulating oxidized phospholipids are markers of developing steatohepatitis temporally distinct from oxidant stress associated with hepatic ethanol catabolism. Previously, circulating markers of the critical transition to pathologic steatohepatitis were unknown. Circulating oxidatively truncated phospholipids are pro-inflammatory and pro-apoptotic mediators with the potential to systemically distribute the effect of chronic ethanol exposure. Suppressing hepatic inflammation, not ethanol catabolism, reduces circulating inflammatory and apoptotic agonists.     

Methyl Sulfone Induces Loss of Metastatic Properties and Reemergence of Normal Phenotypes in a Metastatic Cloudman S-91 (M3) Murine Melanoma Cell Line

Background

The most deadly form of cancer is not lung or colon, breast or prostate; it is any cancer that has become metastatic. Mortality due to metastatic melanoma, one of the most aggressive and deadly cancers, has increased steadily over the last several decades. Unfortunately, the arsenal of chemotherapeutic agents available today is most often unsuccessful at extending and improving the life expectancy of afflicted individuals. We sought to identify an effective and nontoxic agent against metastatic melanoma.

Methodology/Principal Findings

We chose to study Cloudman S-91 mouse melanoma cells (sub-clone M3, CCL53.1) because these cells are highly aggressive and metastatic, representing one of the deadliest types of cancer. Melanoma cells also had an experimental advantage because their morphology, which is easily monitored, relates to the physiology of metastatic cells and normal melanocytes. We chose to test methyl sulfone as a chemotherapeutic agent for two reasons. Because of its chemical structure, we speculated a potential anti-cancer activity by targeting microtubules. Equally important, methyl sulfone has a well-established safety profile in humans. Surprisingly, we found that malignant melanoma cells exposed to methyl sulfone demonstrated the loss of phenotypes characteristic of malignant cells, and the reemergence of phenotypes characteristic of healthy melanocytes. Briefly, over time methyl sulfone induced contact inhibition, loss of ability to migrate through an extracellular matrix, loss of anchorage-independent growth, proper wound healing followed by contact inhibition, irreversible senescence followed by arborization with melanosomes in arbors as seen in normal melanocytes.

Conclusions/Significance

Methyl sulfone may have clinical potential as a non-toxic agent effective against metastatic melanoma. Additionally, methyl sulfone has promise as a tool to explore molecular mechanisms of metastatic transformation as well as fundamental processes such as cell migration, contact inhibition, wound healing and cellular senescence.     

Ab initio prediction of metabolic networks using Fourier transform mass spectrometry data

Fourier transform mass spectrometry has recently been introduced into the field of metabolomics as a technique that enables the mass separation of complex mixtures at very high resolution and with ultra high mass accuracy. Here we show that this enhanced mass accuracy can be exploited to predict large metabolic networks ab initio, based only on the observed metabolites without recourse to predictions based on the literature. The resulting networks are highly information-rich and clearly non-random. They can be used to infer the chemical identity of metabolites and to obtain a global picture of the structure of cellular metabolic networks. This represents the first reconstruction of metabolic networks based on unbiased metabolomic data and offers a breakthrough in the systems-wide analysis of cellular metabolism.     

The use of digit ratios as markers for perinatal androgen action

Since the ratio of the second-to-fourth finger length was first proposed as a marker for prenatal androgen action in 1998, over 100 studies have been published that have either further tested the association between the digit ratio and prenatal androgens, or employed digit ratios as a marker to investigate the association between prenatal androgens and a variety of outcomes, including behavior, fertility, and disease risks. Despite the clear demand for an adult marker of prenatal androgen action and increased use of digit ratios as such a marker, its validity remains controversial. This review (1) evaluates current evidence for the relationship between digit ratios and prenatal androgens (using experimentation with animal models, amniotic testosterone, and congenital adrenal hyperplasia case-control studies), (2) describes opportunities for future validation tests, and (3) compares the potential advantages and disadvantages of digit ratio measures with more established methods for studying the effects of prenatal androgens.     

Fatal hemorrhagic diathesis associated with mild factor IX deficiency in pl/J mice

Surgical implantation of devices into the abdomen of PL/J mice was associated with fatal hemorrhage at 9 to 11 d after surgery. Coagulation profiles were evaluated to determine the underlying cause of this effect. The mean activated partial thromboplastin time (aPTT) of untreated PL/J mice was significantly higher than that of BALB/cByJ and C57BL/6J strains. The addition of human plasmas deficient in factors VIII, XI, or XII, prekallikrein, or high molecular-weight kininogen corrected the elevated aPTT of PL/J mice, but correction did not occur when factor IX-deficient human plasma was added. When compared to an assigned factor IX activity of 100% for pooled plasma from BALB/cByJ mice, C57BL/6J and PL/J mice revealed percent activities of 67% and 16%, respectively. PL/J mice could represent a new model for the study of pathogenesis and therapy of mild factor IX deficiency that is expressed and becomes clinically apparent secondary to major surgery.     

4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites

A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4-hydroxy-2(E)-nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer's disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I-linked and complex II-linked state 3 respirations were inhibited by HNE with IC50 values of approximately 200 microM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N-acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I- and complex II-linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.     

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.