Isolation and Restriction Mapping of a Pseudomonas Plasmid

The effects of the injection of a large amount of N¹ -methylnicotinamide (MNA) (500 mg per kg body weight) on the ratio of N¹ -methyl-4-pyridone-3-carboxamide (4-py) to N¹ -methyl-2-pyridone-5- carboxamide (2-py) excretion, and the activities of 2-py and 4-py forming MNA oxidases were investigated in rats. The injected MN A was excreted very rapidly into the urine; 46 % of the dose was excreted from 0~3hr post-injection, 15% from 3~6hr, 6% from 6~9hr and 1.5% from 9~ 12hr. The ratio of 4-py to 2-py also decreased rapidly; the ratio being about 0.6, 0.4, 0.4 and 0.6 from 0~3hr, 3~6hr, 6~9hr and 9~ 12hr post-injection, respectively. This ratio then recovered rapidly; being about 2, 5.5, 8.5 and 9.7 from 12~24 hr, 24 ~48 hr, 48~72 hr and 72 ~96 hr post-injection, respectively. The normal range of 4-py to 2-py excretion ratio is 8~14. So, this ratio returned to a normal level by day 3 post-injection. The rats were killed 5 hr after the MNA injection. At this time (the lowest ratio was observed around this time), the activities of 2-py and 4-py forming MNA oxidases in the injected group were 59 % and 11 % of the normal levels, respectively. Therefore, it was found that the decreased ratio of 4-py to 2-py excretion with the MNA injection was mainly due to the higher inhibition of the 4-py forming MNA oxidase than of the 2-py forming MNA oxidase by the MNA injection.     

Growth Inhibition by Purine Derivatives and Its Reversal by Pyrimidine Derivatives in a Mutant of Escherichia coli K12

An adenosine-sensitive mutant was isolated from Escherichia coli K12 derivative strain C600. This mutant (designated as PS100) grew slower than parental strain C600in a minimal medium, and its growth was completely inhibited by addition of all kinds of purine bases, nucleosides and nucleotides tested. On the other hand, this growth inhibitory effect of purine derivatives was reversed by co-addition of uridine to the medium. Other pyrimidine derivatives such as uracil, UMP,cytosine, cytidine, CMP and thymidine were also effective for this reversal. The mutant strain, PS100, showed a lower level (7%) of activity for orotate phosphoribosyltransferase than strain C600 did, and accumulated orotic acid in the growth medium. Lysogenization of strain PS100 with λ transducing phage containing the gene for orotate phosphoribosyltransferase (pyrE) resulted in restoration of the activity for orotate phosphoribosyltransferase and removal of growth inhibition by purine derivatives.     

Comprehensive phylogenetic analyses of the Ruppia maritima complex focusing on taxa from the Mediterranean

Recent molecular phylogenetic studies reported high diversity of Ruppia species in the Mediterranean. Multiple taxa, including apparent endemics, are known from that region, however, they have thus far not been exposed to phylogenetic analyses aimed at studying their relationships to taxa from other parts of the world. Here we present a comprehensive phylogenetic analyses of the R. maritima complex using data sets composed of DNA sequences of the plastid genome, the multi-copy nuclear ITS region, and the low-copy nuclear phyB gene with a primary focus on the Mediterranean representatives of the complex. As a result, a new lineage, “Drepanensis”, was identified as the seventh entity of the complex. This lineage is endemic to the Mediterranean. The accessions included in the former “Tetraploid” entity were reclassified into two entities: an Asia–Australia–Europe disjunct “Tetraploid_α” with a paternal “Diploid” origin, and a European “Tetraploid_γ” originating from a maternal “Drepanensis” lineage. Another entity, “Tetraploid_β”, is likely to have been originated as a result of chloroplast capture through backcrossing hybridization between paternal “Tetraploid_α” and maternal “Tetraploid_γ”. Additional discovery of multiple tetraploidizations as well as hybridization and chloroplast capture at the tetraploid level indicated that hybridization has been a significant factor in the diversification of Ruppia.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.     

Freeze-dried platelets promote hepatocyte proliferation in mice

In recent years, platelets are reported to promote liver, as well as bone regeneration and dermal wound healing. Platelets are required not only for thrombocytopenia treating but also for regenerative therapy. Platelets cannot be stored beyond three days, therefore, shortage of fresh platelets sometimes occurs. To preserve platelets for a long duration without degrading growth factors, a freeze-dried technique is required. We report here that platelets can be preserved by freeze-drying, using a programmed freezing method to avoid intracellular ice crystal formation. Freeze-dried platelets kept their morphological countenance and response with the agonist of thrombin was well maintained. Freeze-dried platelets stored adenine nucleotides, PDGF, and IGF-1 the same as those of fresh platelets. Freeze dried platelets also preserved their proliferative effect on hepatocytes identical to that of fresh platelets. These results of our study suggest that freeze dried platelets will obviate the storage problem of fresh platelets.     

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.