Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.     

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Background

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer''s disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and Findings

In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer''s disease mouse model.

Conclusions

Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.     

Neural Mechanisms Influencing Interlimb Coordination during Locomotion in Humans: Presynaptic Modulation of Forearm H-Reflexes during Leg Cycling

Presynaptic inhibition of transmission between Ia afferent terminals and alpha motoneurons (Ia PSI) is a major control mechanism associated with soleus H-reflex modulation during human locomotion. Rhythmic arm cycling suppresses soleus H-reflex amplitude by increasing segmental Ia PSI. There is a reciprocal organization in the human nervous system such that arm cycling modulates H-reflexes in leg muscles and leg cycling modulates H-reflexes in forearm muscles. However, comparatively little is known about mechanisms subserving the effects from leg to arm. Using a conditioning-test (C-T) stimulation paradigm, the purpose of this study was to test the hypothesis that changes in Ia PSI underlie the modulation of H-reflexes in forearm flexor muscles during leg cycling. Subjects performed leg cycling and static activation while H-reflexes were evoked in forearm flexor muscles. H-reflexes were conditioned with either electrical stimuli to the radial nerve (to increase Ia PSI; C-T interval  = 20 ms) or to the superficial radial (SR) nerve (to reduce Ia PSI; C-T interval  = 37–47 ms). While stationary, H-reflex amplitudes were significantly suppressed by radial nerve conditioning and facilitated by SR nerve conditioning. Leg cycling suppressed H-reflex amplitudes and the amount of this suppression was increased with radial nerve conditioning. SR conditioning stimulation removed the suppression of H-reflex amplitude resulting from leg cycling. Interestingly, these effects and interactions on H-reflex amplitudes were observed with subthreshold conditioning stimulus intensities (radial n., ∼0.6×MT; SR n., ∼ perceptual threshold) that did not have clear post synaptic effects. That is, did not evoke reflexes in the surface EMG of forearm flexor muscles. We conclude that the interaction between leg cycling and somatosensory conditioning of forearm H-reflex amplitudes is mediated by modulation of Ia PSI pathways. Overall our results support a conservation of neural control mechanisms between the arms and legs during locomotor behaviors in humans.     

Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis

Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4'-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4'-methoxyisoflavanone, while soybean HID has broader specificity to both 4'-hydroxylated and 4'-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.     

Insulin receptor substrate-1/SHP-2 interaction, a phenotype-dependent switching machinery of insulin-like growth factor-I signaling in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays a role in mutually exclusive processes such as proliferation and differentiation in a variety of cell types. IGF-I is a potent mitogen and motogen for dedifferentiated vascular smooth muscle cells (VSMCs) in vivo and in vitro. However, in differentiated VSMCs, IGF-I is only required for maintaining the differentiated phenotype. Here we investigated the VSMC phenotype-dependent signaling and biological processes triggered by IGF-I. In differentiated VSMCs, IGF-I activated a protein-tyrosine phosphatase, SHP-2, recruited by insulin receptor substrate-1 (IRS-1). The activated SHP-2 then dephosphorylated IRS-1 Tyr(P)-895, resulting in blockade of the pathways from IRS-1/Grb2/Sos to the ERK and p38 MAPK. Conversely, such negative regulation was silent in dedifferentiated VSMCs, where IGF-I activated both MAPKs via IRS-1/Grb2/Sos interaction-linked Ras activation, leading to proliferation and migration. Thus, our present results demonstrate that the IRS-1/SHP-2 interaction acts as a switch controlling VSMC phenotype-dependent IGF-I-induced signaling pathways and biological processes, and this mechanism is likely to be applicable to other cells.     

Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.     

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI.     

Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.     

In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice

We previously reported that deletion of the Fgf2 gene (Fgf2-/-) resulted in decreased bone mass in adult mice. This study examines the effect of haplo-insuffiency (Fgf2+/-) on bone loss in vertebrae from these mutant mice. Fgf2+/+ mice attained peak bone mass at 8-9 months of age. In contrast BMD was significantly reduced in vertebrae from adult (8-9) Fgf2+/- mice. Exogenous FGF-2 rescued reduced bone nodule formation in Fgf2+/- and Fgf2-/- cultures. Runx2 mRNA was reduced in cultures from Fgf2+/- and Fgf2-/- mice. FGF receptor2 mRNA and protein were markedly reduced in Fgf2+/- and Fgf2-/- mice. Decreased bone formation in Fgf2 mutant mice may correlate with impaired FGFR signaling, decreased Runx2 gene expression.