Changes in composition and content of mycolic acids in glutamate-overproducing Corynebacterium glutamicum

Overproduction of glutamate by Corynebacterium glutamicum is induced by biotin limitation or by the supplementation of specific detergents, sublethal amounts of penicillin, or cerulenin. But, it remains unclear why these different treatments, which have different sites of primary action, produce similar effects.In this study, it was found that the cellular content of mycolic acids--characteristic constituents of Corynebacterineae that are synthesized from fatty acids and form a cell surface layer--decreased under all conditions that induced glutamate overproduction. Furthermore, short mycolic acids increased under conditions of biotin limitation and cerulenin supplementation. These results suggest that different treatments produce the same effect that causes defects in the mycolic acid layer. This is perhaps one of the key factors in overproduction of glutamate by C. glutamicum.     

The effects of green tree retention and subsequent prescribed burning on ground beetles (Coleoptera: Carabidae) in boreal pine-dominated forests

We studied how two methods to promote biodiversity in managed forests, i.e. green tree retention and prescribed fire, affect the assemblages of carabid beetles. Our experiment consisted of 24 study sites, each 3–5 ha in size, which had been prepared according to factorial design. Each of the eight treatment combinations determined by the two factors explored – tree retention level (0, 10, 50 m³/ha^?1 and uncut controls) and prescribed use of fire (yes/no) – was replicated three times. We sampled carabids using pitfall traps one year after the treatments. Significantly more individuals were caught in most of the burned sites, but this difference was partially reflective of the trap‐catches of Pterostichus adstrictus. The fire did not increase no. of P. adstrictus in the uncut sites as much as in the other sites. Species richness was significantly affected by both factors, being higher in the burned than in the unburned sites and in the harvested than in the unharvested sites. Many species were concentrated in the groups of retention trees in the burned sites, but only a few were in the unburned sites. The species turnover was greater in the burned than in the unburned sites, as indicated by the NMDS ordinations. Greater numbers of smaller sized species and proportion of brachypterous species were present in the burned sites. Fire‐favored species, and also the majority of other species that prefer open habitats were more abundantly caught in the burned sites than in the unburned sites. Dead wood or logging waste around the traps did not correlate with the occurrence of species. We conclude that carabids are well adapted to disturbances, and that frequent use of prescribed fire is essential for the maintenance of natural assemblages of carabid beetles in the boreal forest. Small retention tree groups can not maintain assemblages of uncut forest, but they can be important by providing food, shelter and breeding sites for many species, particularly in the burned sites.     

Effects of green tree retention and prescribed burning on the crown damage caused by the pine shoot beetles (Tomicus spp.) in pine-dominated timber harvest areas

Abstract: We explored how two recently introduced methods to promote biodiversity during the timber harvest in boreal forests – green tree retention and use of fire – may affect damages caused by pine shoot beetles (Tomicus piniperda and Tomicus minor; Col., Scolytinae) in the surrounding pine forests. The experimental design included 24 separate study sites, 3–5 ha each, which were assigned to different treatments according to factorial design. Retention levels included 0, 10, 50 m³/ha and uncut controls. Twelve of the sites were burned and thus there were three replicates of each treatment combination. Old and new fallen pine shoots were counted from transects in adjacent pine‐dominated forests 2 and 3 years after the treatments. Populations of Tomicus spp. in the sites were monitored using window traps before and after the treatments, and by examining felled sample trees. Numbers of fallen shoots returned to background levels around unburned and burned clearcuts with no retention trees in 3 years after the treatments, but remained still at increased level in burned harvested sites with 10 and 50 m³ of retained trees per hectare. Numbers of fallen shoots in burned uncut forests increased in one site only, where the fire was intense enough to kill large pine trees, but the damage did not extend outside the burned area. Shoot numbers remained at such low levels (<18 000 shoots/ha) in all treatment combinations that growth losses were not likely. Numbers of egg galleries of Tomicus spp. in trees killed by fire were low, indicating that burnings that take place after the swarming of Tomicus beetles create dead wood that is not optimal for the breeding of these pests. We conclude that green tree retention and prescribed use of fire do not automatically affect populations of Tomicus spp. more than traditional forestry operations (thinnings and clearcuttings) do.     

Clinical usefulness of TSAb assay with high polyethylene glycol concentrations.

We previously demonstrated the stimulatory effect of polyethylene glycol (PEG) on thyroid-stimulating antibody (TSAb)-IgG-stimulated cAMP production (thyroid stimulating (TS) index) in porcine thyroid cell (PTC) assay. In the present study the clinical usefulness of the practical method using high PEG concentrations was examined. TS activity using PEG 22.5% precipitated fraction (PF) was significantly higher compared to standard TSAb activity using 12.5% PF from TSAb-positive serum, but the maximum TS activity was observed with PEG 12.5% PF + 4% PEG or PEG 22.5% PF + 2% PEG. In all cases of untreated Graves' patients, TSAb activity determined by PEG 22.5% PF was higher compared to standard TSAb activity using PEG 12. 5% PF from test serum, but the highest TSAb activity was observed by PEG 12.5% PF + 4% PEG without increased cAMP production to normal serum. TSAb was positive in 85% (40/47), 98% (46/47) and 100% (47/47) of untreated Graves' patients by the method of PEG 12.5% PF, PEG 22.5% PF and PEG 12.5% + 4% PEG, respectively. Increased TSAb activity by PEG 12.5% PF + 4% PEG method was also observed even if the standard TSAb activity using PEG 12.5% PF method was negative in the euthyroid states of Graves' patients during antithyroid drug therapy. The stimulatory effect of PEG on TS activity was not found in other thyroidal diseases [thyroiditis chronica (with high serum TSH), thyroid stimulation-blocking antibody (TSBAb)-positive sera (with low serum TSH), adenomatous goiter, subacute thyroiditis, and thyroid cancer]. The stimulatory effect of 5% PEG on TS activity produced directly by small amounts of Graves' serum (50 microl) was also found, although the sensitivity was lower than with PEG-precipitated IgG from 0.2 ml serum. The clinical usefulness of the sensitive TSAb assay using PEG-precipitated IgG or direct serum assay in the presence of high PEG concentrations was demonstrated.     

Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.     

Larval and juvenile fishes occurring with flood tides on an intertidal mudflat in the Tama River estuary, central Japan

Intertidal movements of fish larvae and juveniles on a mudflat in the Tama River estuary, central Japan, were investigated by comparing the abundance and sizes of fishes caught in the intertidal zone during flood tides with those in the subtidal zone during low tides. A total of 28465 individuals, belonging to 9 families and 20 species, were collected by small purse seine. Among the abundant species, planktonic larvae and juveniles of gobiids and Konosirus punctatus were more abundant in the intertidal zone at flood tide than the subtidal zone at low tide. Similar occurrence patterns were found in juvenile Plecoglossus altivelis altivelis and Lateolabrax japonicus, having fully developed swimming abilities. In contrast to these species, much higher abundances of epibenthic juveniles of 2 gobiids (Acanthogobius flavimanus and Gymnogobius macrognathos) were found in the subtidal zone at low tide, although they also utilized the intertidal zone at flood tide.     

Nitrogen resorption from leaves under different growth irradiance in three deciduous woody species

Resorption of nitrogen (N) from senescing leaves is an important conservation mechanism that allows plants to use the same N repeatedly. We measured the extent of N resorption in plants co-occurring in a beech forest to examine the variability of N resorption, especially in relation to growth irradiance. Measurements were done in three deciduous woody species; one adult and several juvenile trees of Fagus crenata and several adult trees of Lindera umbellata and Magnolia salicifolia. N resorption efficiency (R_EFF; percentages of leaf N that is resorbed during leaf senescence) did not differ significantly among leaves under different growth irradiances in any species we studied. R_EFF was affected by the growth stage of the tree in F. crenata with the values being consistently lower in juvenile trees than in the adult tree. N resorption proficiency (R_PROF; N concentration of dead leaves) converged to a similar value in F. crenata juvenile trees and M. salicifolia, irrespective of the presenescent leaf N concentration that was affected by growth irradiance. Again, R_PROF was lower (i.e. absolute N concentration was higher) in juvenile trees than in the adult tree in F. crenata. These results suggest that the growth irradiance does not place a great impact on the extent of N resorption, but the growth stage of the tree is influential in some species. The difference between the adult and juvenile trees may be ascribed to the size of N sink tissues, which is likely to increase with plant age.     

Localization and interaction of the biosynthetic proteins for the lantibiotic, Nukacin ISK-1

Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1. In this study, we characterized NukM and NukT, which are predicted to be involved in modification of prepeptide (NukA) and cleavage of leader peptide and subsequent secretion respectively. Localization analysis of NukM and NukT in the wild-type strain indicated that both proteins were located at the cytoplasm membrane. Interestingly, NukM expressed heterologously in St. carnosus TM300 was also located at the cytoplasm membrane even in the absence of NukT. Yeast two-hybrid assay showed that a complex of at least two each of NukM and NukT was associated with NukA. In vitro interaction analysis by surface plasmon resonance biosensor further suggested that membrane-located NukM interacted with NukA. These results indicate that NukM and NukT form a membrane-located multimeric protein complex and that post-translational modification of nukacin ISK-1 would occur at the cytoplasm membrane.     

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies . Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.