Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors

Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.     

Characteristics of Resonance in Heart Rate Variability Stimulated by Biofeedback

As we previously reported, resonant frequency heart rate variability biofeedback increases baroreflex gain and peak expiratory flow in healthy individuals and has positive effects in treatment of asthma patients. Biofeedback readily produces large oscillations in heart rate, blood pressure, vascular tone, and pulse amplitude via paced breathing at the specific natural resonant frequency of the cardiovascular system for each individual. This paper describes how resonance properties of the cardiovascular system mediate the effects of heart rate variability biofeedback. There is evidence that resonant oscillations can train autonomic reflexes to provide therapeutic effect. The paper is based on studies described in previous papers. Here, we discuss the origin of the resonance phenomenon, describe our procedure for determining an individual's resonant frequency, and report data from 32 adult asthma patients and 24 healthy adult subjects, showing a negative relationship between resonant frequency and height, and a lower resonant frequency in men than women, but no relationship between resonant frequency and age, weight, or presence of asthma. Resonant frequency remains constant across 10 sessions of biofeedback training. It appears to be related to blood volume.     

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-snRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-snRNP reconstructed to approximately 2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 snRNPs, show how U4/U6 and U5 combine to form tri-snRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-snRNP. We suggest that this central tri-snRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.     

The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group

A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of Guillain-Barré syndrome, had unidentified labile, capsular polysaccharide (CPS) structures. In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups. Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-beta-D-GlcA6NGro-(1-3)-beta-D-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol. A labile alpha-sorbofuranose branch located at C2 of GlcA was determined to have the L configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan. A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group. Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain. These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes. The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C. jejuni.     

Complete Mitochondrial Genome and Phylogeny of Pleistocene Mammoth Mammuthus primigenius

Phylogenetic relationships between the extinct woolly mammoth(Mammuthus primigenius), and the Asian(Elephas maximus) and African savanna(Loxodonta africana) elephants remain unresolved. Here, we report the sequence of the complete mitochondrial genome (16,842 base pairs) of a woolly mammoth extracted from permafrost-preserved remains from the Pleistocene epoch—the oldest mitochondrial genome sequence determined to date. We demonstrate that well-preserved mitochondrial genome fragments, as long as ~1,600–1700 base pairs, can be retrieved from pre-Holocene remains of an extinct species. Phylogenetic reconstruction of the Elephantinae clade suggests thatM. primigenius andE. maximus are sister species that diverged soon after their common ancestor split from theL. africana lineage. Low nucleotide diversity found between independently determined mitochondrial genomic sequences of woolly mammoths separated geographically and in time suggests that north-eastern Siberia was occupied by a relatively homogeneous population ofM. primigenius throughout the late Pleistocene.     

Semisynthesis and pharmacological activities of Tetrac analogs: Angiogenesis modulators

Novel Tetrac analogs were synthesized and then tested. Anti-angiogenesis efficacy was carried out using the Chick Chorioallantoic Membrane (CAM) model and the mouse matrigel model for angiogenesis. Pharmacological activities showed Tetrac can accommodate numerous modifications and maintain anti-angiogenesis activity.     

<Emphasis Type="Italic">Bathyphellia margaritacea</Emphasis> (Cnidaria: Actiniaria): the most northern species of the world

The sea anemone Bathyphellia margaritacea (Danielssen in Actinida. The Norwegian North-Atlantic expedition 1876–1878, Groendahl, Oslo, 1890) was collected by the research submersible MIR at the North Pole at a depth of 4,262 m and by the North Pole Drifting Station NP-22 in the American sector of Arctic Ocean covered by permanent ice. These widely separated records significantly increase the known geographic range of the species. B. margaritacea is highly plastic and has an ability to occupy different types of substrates. It appears to be the only species of sea anemone that is able to range in the high Arctic up to the North Pole and the only reliably identified species known from this part of the world.     

Glutamate uptake determines pathway specificity of long-term potentiation in the neural circuitry of fear conditioning

Long-term synaptic modifications in afferent inputs to the amygdala underlie fear conditioning in animals. Fear conditioning to a single sensory modality does not generalize to other cues, implying that synaptic modifications in fear conditioning pathways are input specific. The mechanisms of pathway specificity of long-term potentiation (LTP) are poorly understood. Here we show that inhibition of glutamate transporters leads to the loss of input specificity of LTP in the amygdala slices, as assessed by monitoring synaptic responses at two independent inputs converging on a single postsynaptic neuron. Diffusion of glutamate ("spillover") from stimulated synapses, paired with postsynaptic depolarization, is sufficient to induce LTP in the heterosynaptic pathway, whereas an enzymatic glutamate scavenger abolishes this effect. These results establish active glutamate uptake as a crucial mechanism maintaining the pathway specificity of LTP in the neural circuitry of fear conditioning.     

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.