Spin trapping of the superoxide anion: complications in the use of the water-soluble nitroso-aromatic reagent DBNBS

Sodium 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) is reported to be a useful spin trap for the measurement of superoxide anions in aqueous solution. However, the signal observed arises from interaction of the spin trap with some species other than the superoxide radical or hydrogen peroxide, a product of its dismutation, as the addition of both superoxide dismutase and catalase to a superoxide generating system failed to attenuate the signal. Therefore caution must be employed in the interpretation of results obtained using this spin trap.     

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.     

The Apectodinium acme and terrestrial discharge during the Paleocene-Eocene thermal maximum: new palynological, geochemical and calcareous nannoplankton observations at Tawanui, New Zealand

Manifestations of profound perturbations in biogeochemical systems during the Paleocene-Eocene thermal maximum (PETM) include a prominent global negative δ¹³C and a pronounced increase in the relative abundance of dinoflagellate cysts (dinocysts) assigned to the genus Apectodinium. While motile representatives of Apectodinium were most likely thermophilic and heterotrophic, the underlying causes of this dinoflagellate response are not well understood. Here we provide new insight by examining the palynology, chemistry and calcareous nannoplankton across the PETM in a continental slope section at Tawanui, New Zealand. Across the PETM, marked changes in the relative abundance of Apectodinium vary antithetically with significant changes in the δ¹³C of carbonate and organic matter. In general, the high relative abundance of Apectodinium relates to enhanced concentrations of dinocysts, signifying a ‘bloom’ of Apectodinium in surface waters during the PETM. Changes in Apectodinium and δ¹³C records correspond to variations in many other parameters, including a smaller negative shift in bulk carbonate δ¹³C than expected, increased terrestrial palynomorphs, elevated TOC and C/N ratios, lower carbonate contents, higher SiO₂ and Al₂O₃ contents, and lower Si/Al ratios. All of these variations can be explained by an increase in delivery of terrigenous material to the continental margin. A peak in the relative abundance of Glaphyrocysta dinocysts at the onset of the PETM may indicate greater down slope transport of neritic material. Changes in calcareous nannoplankton abundances suggest increased nutrient availability in surface waters during the PETM. The combined results show that Apectodinium-dominated assemblages, global perturbations in carbon isotopes and enhanced terrigenous delivery closely correspond in time at Tawanui. A sudden and massive carbon injection to the ocean-atmosphere system may have enhanced weathering and increased terrigenous inputs to continental margins during the PETM. We further suggest that these inputs caused the Apectodinium acme by elevating primary productivity in marginal seas.     

Increased Zinc and Manganese in Parallel with Neurodegeneration,Synaptic Protein Changes and Activation of Akt/GSK3 Signaling in Ovine CLN6 Neuronal Ceroid Lipofuscinosis

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.     

Studies on the mechanism of acetaldehyde-mediated inhibition of aspartate aminotransferase (GOT) in human erythrocytes

The mechanism of acetaldehyde-mediated GOT inhibition was studied in human red cells. In the GOT assay mix, acetaldehyde competitively inhibits activation of apoGOT by pyridoxal phosphate and pyridoxamine phosphate. However, Ki values are 100-1000 times greater than Km values for these B6 vitamers. Moreover, incubation of undiluted lysates with acetaldehyde at 37 degrees inhibits GOT activity without increasing apoGOT levels and without altering affinity of apoGOT for either B6 coenzyme. In undiluted lysates, inhibition is not prevented by disulfiram. However, incubation at 4 degrees prevents both acetaldehyde metabolism and GOT inhibition while preincubation with NaF prevents GOT inhibition without affecting acetaldehyde disappearance. The effect of NaF is completely reversed by pyruvate but only partially reversed by NADH. Glyceraldehyde 3-phosphate, the only glycolytic intermediate which directly inhibits GOT, does not reverse the NaF effect. Thus, inhibition of GOT by acetaldehyde (a) requires nonoxidative metabolism of acetaldehyde and (b) is not mediated either by glycolytic substrates or by impaired binding of B6 vitamers to the GOT apoenzyme. Since NaF had no effect on a lysate deficient in glucose 6-phosphate dehydrogenase, the hexose monophosphate shunt may play a role in acetaldehyde-mediated GOT inhibition.     

Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods

We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.     

A Unique Sugar-binding Site Mediates the Distinct Anti-influenza Activity of Pig Surfactant Protein D

Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.