Prey selection by a spider wasp,Batozonellus lacerticida (Hymenoptera: Pompilidae): Effects of seasonal variation in prey species,size and density

Prey selection by a spider wasp,Batozonellus lacerticida, was investigated at the riverside of the Toyohira River in Misumai, Sapporo, Hokkaido over 7 years from 1981 to 1987. Seventeen species of araneid spiders were found in the study area and six of them were hunted by the wasps. Most (97.3%) of the 223 prey records obtained were of three species,Araneus macacus, A. marmoreus andA. pinguis. Nesting activity of the wasps continued until mid-August in 1981, 1982 and 1986, but stopped in July in 1984, 1985 and 1987. The length of nesting period was related to whether the wasps successfully switched prey species fromA. macacus toA. marmoreus andA. pinguis andA. pinguis or not Switching seemed to occur at the phase when density ofA. macacus remained high, depending on the density of largerA. marmorus andA. pinguis. This switching had large effects on species composition and size distribution of actual prey. Chesson's index α calculated at every 10 day period revealed that the wasps preferredA. macacus to the two other species and large prey to medium and small ones. The effect of prey density on preference depended on whetherA. macacus was present or absent. In the presence of it there were only slight differences in preference among various conditions of prey density. However, whenA. macacus was absent, densities of bothA. marmoreus andA. pinguis had some complementary effects on preference. The importance of prey size selection by pompilid wasps, and implications of density effects on preference are discussed.     

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.     

Formation of circular polyribosomes in wheat germ cell-free protein synthesis system

We have compared the urea stability of the human aromatic amino acid hydroxylases (AAAHs), key enzymes involved in neurotransmitter biosynthesis and amino acid homeostasis. Tyrosine-, tryptophan- and phenylalanine hydroxylase (TH, TPH and PAH, respectively) were transiently activated at low urea concentrations and rapidly inactivated in >3 M urea. The denaturation of TH occurred through two cooperative transitions, with denaturation midpoints of 1.41+/-0.06 and 5.13+/-0.05 M urea, respectively. Partially denatured human TH (hTH) retained more of its secondary structure than human PAH (hPAH), and was found to exist as tetramers, whereas hPAH dissociated into dimers. Furthermore, the urea-induced aggregation of hPAH was 100-fold higher than for hTH. These results suggest that the denatured state properties of the AAAHs contribute significantly to the stability of these enzymes and their tolerance towards missense mutations.     

Restoration of contralateral representation in the mouse somatosensory cortex after crossing nerve transfer

Avulsion of spinal nerve roots in the brachial plexus (BP) can be repaired by crossing nerve transfer via a nerve graft to connect injured nerve ends to the BP contralateral to the lesioned side. Sensory recovery in these patients suggests that the contralateral primary somatosensory cortex (S1) is activated by afferent inputs that bypassed to the contralateral BP. To confirm this hypothesis, the present study visualized cortical activity after crossing nerve transfer in mice through the use of transcranial flavoprotein fluorescence imaging. In naïve mice, vibratory stimuli applied to the forepaw elicited localized fluorescence responses in the S1 contralateral to the stimulated side, with almost no activity in the ipsilateral S1. Four weeks after crossing nerve transfer, forepaw stimulation in the injured and repaired side resulted in cortical responses only in the S1 ipsilateral to the stimulated side. At eight weeks after crossing nerve transfer, forepaw stimulation resulted in S1 cortical responses of both hemispheres. These cortical responses were abolished by cutting the nerve graft used for repair. Exposure of the ipsilateral S1 to blue laser light suppressed cortical responses in the ipsilateral S1, as well as in the contralateral S1, suggesting that ipsilateral responses propagated to the contralateral S1 via cortico-cortical pathways. Direct high-frequency stimulation of the ipsilateral S1 in combination with forepaw stimulation acutely induced S1 bilateral cortical representation of the forepaw area in naïve mice. Cortical responses in the contralateral S1 after crossing nerve transfer were reduced in cortex-restricted heterotypic GluN1 (NMDAR1) knockout mice. Functional bilateral cortical representation was not clearly observed in genetically manipulated mice with impaired cortico-cortical pathways between S1 of both hemispheres. Taken together, these findings strongly suggest that activity-dependent potentiation of cortico-cortical pathways has a critical role for sensory recovery in patients after crossing nerve transfer.     

Large-scale production of N-acetyllactosamine through bacterial coupling.

A large-scale production system of N-acetyllactosamine, a core structure of various oligosaccharides, was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains over-expressed the UDP-Gal biosynthetic genes and the beta-(1-->4)-galactosyltransferase gene of Neisseria gonorrhoeae, respectively. C. ammoniagenes contributed the production of UTP from orotic acid. N-Acetyllactosamine was accumulated at 279 mM (107 g L-1) after a 38 h reaction (2.5 L in volume) starting from orotic acid, D-galactose, and 2-acetamido-2-deoxy-D-glucose.     

Contribution of the exosome-associated form of secreted endoplasmic reticulum aminopeptidase 1 to exosome-mediated macrophage activation

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages.     

Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130(Cas), but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1-induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1-induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1-induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1-induced human aortic endothelial cells migration.     

Relationships among Mercury Concentration,and Stable Isotope Ratios of Carbon and Nitrogen in the Scalp Hair of Residents from Seven Countries: Effects of Marine Fish and C4 Plants Consumption

We analyzed the Hg concentration, and δ¹³C and δ¹⁵N values in the scalp hair of residents from seven countries; Vietnam, New Zealand, Spain, the USA, South Korea, Brazil and Japan. Relationships among the data in each country and among the seven countries were then examined. The highest Hg concentration as well as the highest or higher δ¹⁵N value in each country was found in the hair of a heavy marine fish-eater, whereas the lowest Hg concentration and δ¹⁵N value were found in the hair of a vegetarian or non (marginal)-fish eater. Hg concentrations were positively correlated with the δ¹⁵N values in each country, and increased markedly in samples with δ¹⁵N values exceeding 9.0 ‰, probably due to fish consumption. The highest Hg concentration could be found in sample, with a δ¹³C value between -19 and -18‰, probably reflecting the δ¹³C value of the marine food web.     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.