In vitro and in vivo transferrable beta-lactam resistance due to a new plasmid-mediated oxyiminocephalosporinase from a clinical isolate of Proteus mirabilis

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.     

Immunological responses of hamsters in the acquired immune state to Mycoplasma pneumoniae infection

Protective effects of vaccination of hamsters against Mycoplasma pneumoniae infection, evaluated according to the recovery of mycoplasmas and histopathological changes in the respiratory tract after challenge infection, persisted for at least 6 months after the final vaccination. Serum antibody levels reached a maximum in the second week after the last vaccination and decreased markedly between the first and the third months, but increased again in sera obtained from animals given booster injections. Metabolism-inhibiting antibodies were detected in bronchial washings of animals showing high resistance obtained by vaccinal or passive immunization. Antiserum transfer was also effective for protection but cell-mediated immune responses were not demonstrated in any animals up to 6 months after the vaccination. Even after 10 months, suppression of both mycoplasmal proliferation and lung lesions was apparent, and a single dose of the vaccine induced a significant booster effect. These findings suggest that (1) humoral immunity is more important than cell-mediated immunity in resistance of hamsters to M. pneumoniae pneumonia, and (2) the antibody secreted in the respiratory tract may be involved in the local defense mechanisms.     

Canine trachealis muscle shortening and cartilage mechanics.

Canine trachealis muscle will shorten by 70% of resting length when maximally stimulated in vitro. In contrast, trachealis muscle will shorten by only 30-40% when stimulated in vivo. To examine the possibility that an elastic load applied by the tracheal cartilage contributes to the in vivo limitation of shortening, single pairs of sonomicrometry crystals were inserted into the trachealis muscle at the level of the fifth cartilage ring in five dogs. The segment containing the crystals was then excised and mounted on a tension-testing apparatus. Points on the active length-tension curve and the passive length-tension relation of the cartilage only were determined. The preload applied to the muscle before contraction varied from 10 to 40 g (mean 21 +/- 4 g). The afterload applied by the cartilage during trachealis contraction ranged from 13 to 56 g (30 +/- 6 g). The calculated elastic afterloads were substantial and appeared to be sufficient to explain the degree of shortening observed in four of the seven rings; in the remaining three rings, the limitation of shortening was greater than would be expected from the elastic load provided by the cartilage. Additional sources of loading and/or additional mechanisms may contribute to limited in situ shortening. In summary, tracheal cartilage applies a preload and an elastic afterload to the trachealis that are substantial and contribute to the limitation of trachealis muscle shortening in vivo.     

Missing piece of top predator-based conservation: Demographic analysis of an owl population on a remote subtropical island

Top predators are frequently the target of conservation programs. Owls are such predators. However, previous studies of owls are biased to species occurring in temperate regions, whereas most owl species occur in tropical or subtropical regions and are understudied. Furthermore, owls are often endemic to islands and of unknown conservation status. Demographic data for such species are especially scarce although they are essential for initiating and promoting their conservation. As a case study of demographic analysis of owls in a tropical or subtropical area and on islands, we applied an integrated population model to 7-year monitoring data (2012–2018) of the Ryukyu Scops Owl population on Minami-daito Island, Japan. We used survival history data from 903 individuals, reproduction and sex ratio data from 213 broods, and count data of 2,526 individuals in total. Long-term averages of annual survival rates were 0.73 for adult females and 0.74 for adult males, although the sexual difference was not significant. Sex ratio estimates fluctuated annually and long-term averages were slightly skewed to males: 0.51 among fledglings, 0.54 among yearlings, and 0.52 among adults. Long-term averages of population size were estimated to be 273.4 females and 296.8 males. The long-term average of population growth rate was 0.98, suggesting a slightly declining trend. It was fortunate to recognize the declining trend during the early phase. Considering the general lack of fundamental ecological data on owls of tropical or subtropical areas and on islands, it seems likely that many endangered owl populations await conservation efforts.     

Characteristics of microbial fermentation and potential digestibility of fiber in the hindgut of dugongs (Dugong dugon)

The number of microorganisms in the hindgut of dugongs (Dugong dugon) were estimated and their in vitro volatile fatty acid (VFA) production and degradation of eelgrass measured. Scanning electron microscopy showed that some rod bacteria attached to the surface of plant tissue degraded and eroded the cell walls. Number of starch-, lactate-, cellobiose-, pectin-, xylan- and cellulose-utilizing bacteria, sulfate-reducing bacteria and methane-producing bacteria were estimated at 10⁹ ～ 10¹⁰ colony forming units g^?1. Microorganisms degraded the cellulose and noncellulolytic components of the eelgrass, and about 47.3% of dry matter was degraded after 36?h in vitro incubation. The total VFA concentration was 10.5?mmol?dL^?1 at 36?h incubation, which included 55.7?mol% acetate, 18.0?mol% n-butyrate and 15.1?mol% propionate. The gas composition of in vitro fermentation was 68.4% carbon dioxide, 22.2% methane and 9.4% hydrogen.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Stability of cytochromes c′ from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure

Extremophiles - The stability of dimeric cytochrome c′ from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the...     

Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism