Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO₂⁻) (30%), nitrous oxide (N₂O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [¹⁵N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N₂O with two molecular mass ions: one at 44 Da, corresponding to ¹⁴N¹⁴NO, and the second at 45 Da, corresponding to ¹⁵N¹⁴NO. The nonlabeled N₂O could be formed only from -NO₂, whereas the ¹⁵N-labeled one was presumed to originate from a nitramine group (¹⁵N-¹⁴NO₂) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C₂H₅N₃O₃. When the experiment was repeated with ring-labeled [¹⁵N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-¹⁴C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in ¹⁴CO₂ (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO₂ and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Obesity polymorphisms identified in genome-wide association studies interact with n-3 polyunsaturated fatty acid intake and modify the genetic association with adiposity phenotypes in Yup’ik people

n-3 Polyunsaturated fatty acids (n-3 PUFAs) have anti-obesity effects that may modulate risk of obesity, in part, through interactions with genetic factors. Genome-wide association studies (GWAS) have identified genetic variants associated with body mass index (BMI); however, the extent to which these variants influence adiposity through interactions with n-3 PUFAs remains unknown. We evaluated 10 highly replicated obesity GWAS single nucleotide polymorphisms (SNPs) for individual and cumulative associations with adiposity phenotypes in a cross-sectional sample of Yup’ik people (n = 1,073) and evaluated whether genetic associations with obesity were modulated by n-3 PUFA intake. A genetic risk score (GRS) was calculated by adding the BMI-increasing alleles across all 10 SNPs. Dietary intake of n-3 PUFAs was estimated using nitrogen stable isotope ratio (δ¹⁵N) of red blood cells, and genotype–phenotype analyses were tested in linear models accounting for familial correlations. GRS was positively associated with BMI (p = 0.012), PBF (p = 0.022), ThC (p = 0.025), and waist circumference (p = 0.038). The variance in adiposity phenotypes explained by the GRS included BMI (0.7 %), PBF (0.3 %), ThC (0.7 %), and WC (0.5 %). GRS interactions with n-3 PUFAs modified the association with adiposity and accounted for more than twice the phenotypic variation (~1–2 %), relative to GRS associations alone. Obesity GWAS SNPs contribute to adiposity in this study population of Yup’ik people and interactions with n-3 PUFA intake potentiated the risk of fat accumulation among individuals with high obesity GRS. These data suggest the anti-obesity effects of n-3 PUFAs among Yup’ik people may, in part, be dependent upon an individual’s genetic predisposition to obesity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0340-z) contains supplementary material, which is available to authorized users.     

Acute Administration of Non-Classical Estrogen Receptor Agonists Attenuates Ischemia-Induced Hippocampal Neuron Loss in Middle-Aged Female Rats

Background

Pretreatment with 17β-estradiol (E2) is profoundly neuroprotective in young animals subjected to focal and global ischemia. However, whether E2 retains its neuroprotective efficacy in aging animals, especially when administered after brain insult, is largely unknown.

Methodology/Principal Findings

We examined the neuroprotective effects of E2 and two agonists that bind to non-classical estrogen receptors, G1 and STX, when administered after ischemia in middle-aged rats after prolonged ovarian hormone withdrawal. Eight weeks after ovariectomy, middle-aged female rats underwent 10 minutes of global ischemia by four vessel occlusion. Immediately after reperfusion, animals received a single infusion of either E2 (2.25 µg), G1 (50 µg) or STX (50 µg) into the lateral ventricle (ICV) or a single systemic injection of E2 (100 µg/kg). Surviving pyramidal neurons in the hippocampal CA1 were quantified 1 week later. E2 and both agonists that target non-classical estrogen receptors (G1 and STX) administered ICV at the time of reperfusion provided significant levels of neuroprotection, with 55–60% of CA1 neurons surviving vs 15% survival in controls. A single systemic injection of a pharmacological dose of E2 also rescued approximately 50% of CA1 pyramidal neurons destined to die. To determine if E2 and G1 have similar mechanisms of action in hippocampal neurons, we compared the ability of E2 and G1 to modify CA1 pyramidal neuron responses to excitatory inputs from the Schaffer collaterals recorded in hippocampal slices derived from female rats not subjected to global ischemia. E2 and G1 (10 nM) significantly potentiated pyramidal neuron responses to excitatory inputs when applied to hippocampal slices.

Conclusions/Significance

These findings suggest (1) that middle-aged female rats retain their responsiveness to E2 even after a long period of hormone withdrawal, (2) that non-classical estrogen receptors may mediate the neuroprotective actions of E2 when given after ischemia, and (3) that the neuroprotective efficacy of estrogens may be related to their modulation of synaptic activity in hippocampal slices.     

Relationships between permeable vessels, nerves, and mast cells in rat cutaneous neurogenic inflammation

Electrical stimulation of rat sensory nerves produces cutaneous vasodilation and plasma protein extravasation, a phenomenon termed "neurogenic inflammation". Rat skin on the dorsum of the paw developed neurogenic inflammation after electrical stimulation of the saphenous nerve. In tissue sections, the extravasation of the supravital dye monastral blue B identified permeable vessels. Mast cells were identified by toluidine blue stain. Permeable vessels were significantly more dense in the superficial 120 microns of the dermis than in the deeper dermis, whereas mast cells were significantly more frequent in the deeper dermis. The relationships between nociceptive sensory nerve fibers, permeable vessels, and mast cells were examined by indirect immunohistochemistry for calcitonin gene-related peptide (CGRP), neurokinin A (NKA), and substance P (SP). CGRP-, NKA-, and SP-containing nerves densely innervated the superficial dermis and appeared to innervate the vessels that became permeable during neurogenic inflammation. In contrast, mast cells were not associated with either permeable vessels or nerve fibers. These data suggest that electrical stimulation of rat sensory nerves produces vascular permeability by inducing the release of neuropeptides that may directly stimulate the superficial vascular bed. Mast cells may not be involved in this stage of cutaneous neurogenic inflammation in rat skin.     

Vinflunine, a novel microtubule inhibitor, suppresses calmodulin interaction with the microtubule-associated protein STOP

Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM-target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+-CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+-CaM-STOP complex formation. Our results revealed different binding modes to Ca2+-CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.     

Photoreceptor membrane turnover in the crayfish Cherax destructor: electron microscopy and anti-rhodopsin electron-microscopic immunocytochemistry

Summary Examination of the ultrastructure of retinula cells of the Australian crayfish Cherax destructor at different times over a 24-hour cycle, together with patterns of anti-rhodopsin antigenicity, has lead to the formulation of a model of photoreceptor membrane turnover in these animals. Its main features are: (a) the existence of two bursts of rhabdomeral membrane breakdown; one, light-sensitive and synchronous, occurring at dawn, the other, constituting the first part of the membrane replacement phase itself, occurring during the afternoon and night, (b) the desynchronisation of the replacement phase of turnover between animals and to a lesser extent between cells of the same retina, (c) confinement of ultrastructurally detectable signs of photoreceptor membrane processing to the retinula cells themselves, and (d) replacement of a substantial part if not all of the rhabdomeral membrane daily. This model is compatible with many of the observations reported on the American crayfish Procambarus, and utilises the same basic mechanisms that are believed to operate in photoreceptor membrane turnover in many other arthropod compound eyes.     

Characterization of Potocki-Lupski syndrome (dup(17)(p11.2p11.2)) and delineation of a dosage-sensitive critical interval that can convey an autism phenotype

The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal microduplication syndrome described--the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (~3.7 Mb), 13 subjects (~37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.     

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.     

Genome Sequence of Lactobacillus mucosae LM1, Isolated from Piglet Feces

Lactobacillus mucosae LM1, isolated from stool samples of a healthy piglet, displays good in vitro mucin adhesion and antimicrobial activity against pathogenic bacteria. To elucidate its antimicrobial effects and to find its epithelial cell and mucin adhesion genes, the genomic sequence of L. mucosae LM1 was investigated.