The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

The oxo/peroxo debate: a nonheme iron perspective

The oxygen activation mechanisms proposed for nonheme iron systems generally follow the heme paradigm in invoking the involvement of iron-peroxo and iron-oxo species in their catalytic cycles. However, the nonheme ligand environments allow for end-on and side-on dioxygen coordination and impart greater flexibility in the modes of dioxygen activation. The currently available evidence for nonheme iron-peroxo and iron-oxo intermediates is summarized and discussed in light of the ongoing discussion on the nature of the oxidant(s) in heme enzymes.     

Ascaris lumbricoides: succinate and tiglate in hemolymph

When hemolymph is taken from Ascaris lumbricoides at the time the worm is collected from pigs, it contains acetic, propionic, 2-methylbutyric, n-valeric, 2-methylvaleric, and succinic acid radicals; tiglic acid is absent.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

Characterization of tubule and monomer derived from VP4 protein of infectious bursal disease virus

The VP4 protein of infectious bursal disease virus (IBDV) is a serine protease that processes the polyprotein for viral assembly. VP4 has been found to associate primarily with type II IBDV tubules that are 24 nm in diameter. In this study, a chimeric VP4, assigned as HS1VP4, was constructed with a VP4-autocleavage site inserted between the N-terminal His-tag and the VP4 sequence. The results showed that the VP4 forms tubules after the self-cleavage of HS1VP4 when expressed in Escherichia coli. Furthermore, a deletion of 28 amino acids at the C-terminus of VP4 resulted in monomers and dimers instead of tubule formation; mutants of S652A and K692A at active site destroyed the activity. The endopeptidase activity of these monomers and dimers was approximately 12.5 times higher than that of VP4 tubules. Additionally, the formation of tubules inhibited VP4 protease activity, as demonstrated through in vitro assays. The production and characterization of monomers or dimers that have greater endopeptidase activity and protease activity than tubules can provide further insight into VP4 tubule assembly and the regulation of VP4 activity in host cells; this insight will facilitate the development of new anti-IBDV strategies.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Heart design: free ADP scales with absolute mitochondrial and myofibrillar volumes from mouse to human

Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005±50 (n=16), 460±32 (n=10) and 330±22 (n=5) mM⁻¹ and a free cytosolic [ADP] concentration of 13, 18 and 22 μM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (−0.28, r=0.99 and −0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=−0.26, r=0.99) and myofibrillar volumes (slope=−0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.     

Sri Lankan passion fruit mottle virus, a potyvirus infecting golden passion fruit in Sri Lanka

A sap-transmissible virus, provisionally named Sri Lankan passion fruit mottle virus (SLPFMV), was isolated from Passiflora edulis f. flavicarpa and shown to induce leaf mottling and distortion in that host. The virus infected 23 species in five plant families with systemic infection being common in the Passifloraceae. Chenopodium amaranticolor was a good local lesion host and Passiflora foetida was a useful systemic host for purification. In P. foetida extracts, SLPFMV lost infectivity after 10 min between 70–75°C, 6–7 days at 20–23°C and at dilutions of 10^--⁵ -W^-6. The virus had flexuous, filamentous particles with a normal length of c. 841 nm. Two polypeptides of mol. wt c. 33 200 and 28 700 were detected in purified virus preparations, and a major species of double-stranded RNA (mol. wt 7.0 × 10⁶), was detected in infected plants. Pinwheels, tubular and laminated inclusions were found in ultrathin sections of infected P. edulis f. plavicurpa and cylindrical inclusions were observed in epidermal strips. SLPFMV was transmitted by the aphids Myzus persicae, Aphis spiraecola, A. gossypü and A. cruccivora after brief acquisition feeds. SLPFMV reacted with antisera to several potyviruses including passion fruit woodiness virus, passion fruit ringspot virus, potato virus Y and watermelon mosaic virus 2 and thus, apparently, is a member of the potyvirus group.     

Localization of vanadium-containing particles in the lungs of uranium/vanadium miners

Several geological formations of the Utah-Colorado mining region mined for uranium ore during and after World War II had been mined earlier for vanadium. Therefore, most miners and millers from that region were exposed to those metals’ ores or tailings at one time or another. Preliminary investigation to determine uranium and vanadium retained in the lungs of a former uranium miner and miller from this region, who died of lung cancer (mesothelioma), showed a high nonuniform distribution of vanadium. This observation led to the hypothesis that the vanadium content in the lungs could be associated with inhaled particles. Further examination of spectra of characteristic X-rays obtained by scanning particle-induced X-ray emission (microPIXE) of an autopsy sample of this lung indicated that vanadium was indeed present in localized sites within the 20-μm spatial resolution of the proton beam. This work points out that the microPIXE-RBS (Rutherford backscattering) test for vanadium can be used for site localization of inhaled particles retained in the lungs. Further studies are in progress to: (i) locate uranium-bearing particles in lung tissues of former uranium miners and millers; and (ii) evaluate the local doses of alpha radiation received from these particles.