Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

Immune responses to Puumala virus infection and the pathogenesis of nephropathia epidemica

Puumala virus, causative agent of a mild form of hemorrhagic fever with renal syndrome, also known as nephropathia epidemica, induces long-lasting humoral and cellular immunity in patients. The virus itself is not cytopathic, and the immune responses to the virus may be involved in teh pathogenesis of the disease.     

Genetic divergence between and within two color morphotypes of <Emphasis Type="Italic">Parapercis sexfasciata</Emphasis> (Perciformes: Pinguipedidae) from Tosa Bay,southern Japan

The anterior half of the mitochondrial DNA control region (mtCR) sequence (ca. 400 base pairs) was compared between two color morphotypes (A, B) of Parapercis sexfasciata from Tosa Bay, southern Japan, using 16 and 21 specimens, respectively. Intramorphotypic mtCR divergences were only 0.0–0.5% and 1.0–2.5% for morphotypes A and B, respectively. In contrast, intermorphotypic mtCR divergence was much greater, 12.7–14.0%. Furthermore, phylogenetic analysis using a neighbor-joining algorithm, with P. multifasciata as an outgroup, showed that each morphotype was reciprocally monophyletic. These results and the distinct coloration and overlapping distribution indicate that the two color morphotypes of P. sexfasciata represent two distinct species. Mismatch distribution analysis suggested that both morphotypes had undergone population expansion; however, estimates of initial population sizes and mutational timescales suggested that morphotype B comprises historically larger and older populations than morphotype A.     

A new hypothesis for species coexistence: male–male repulsion promotes coexistence of competing species

We propose a new hypothesis for species coexistence by considering behavioral interactions between individuals. The hypothesis states that repulsive behavior between conspecific males (male–male repulsion) creates space for competing species, which promotes their coexistence. This hypothesis can explain the coexistence of two competing species even when their ecological niches completely overlap in spatially homogeneous environments. In addition, the mechanisms underlying such behavior might play a role in enabling the coexistence of two species immediately after speciation, with little or no niche differentiation, as in the case of cichlid fish communities, for example. Although there is limited evidence supporting this hypothesis, it can nevertheless explain the occurrence of species coexistence and biodiversity, which cannot be explained by previous theories.     

A rapid biosensor chip assay for measuring of telomerase activity using surface plasmon resonance

Considerable interest has been focused on telomerase because of its potential use in assays for cancer diagnosis, and for anti-telomerase drugs as a strategy for cancer chemotherapy. A number of assays based on the polymerase chain reaction (PCR) have been developed for evaluation of telomerase activity. To overcome the disadvantages of the conventional telomerase assay [telomeric repeat amplification protocol (TRAP)] related to PCR artifacts and troublesome post-PCR procedures, we have developed a telomeric repeat elongation (TRE) assay which directly measures telomerase activity as the telomeric elongation rate by biosensor technology using surface plasmon resonance (SPR). 5′-Biotinylated oligomers containing telomeric repeats were immobilized on streptavidin-pretreated dextran sensor surfaces in situ using the BIACORE apparatus. Subsequently, the oligomers associated with the telomerase extracts were elongated in the BIACORE apparatus. The rate of TRE was calculated by measuring the SPR signals. We examined elongation rates by the TRE assay in 18 cancer and three normal human fibroblast cell lines, and 12 human primary carcinomas and matching normal tissues. The elongation rates increased in a concentration- and time-dependent manner. Those of cancer cells were two to 10 times higher than fibroblast cell lines and normal tissues. Telomerase activities and its inhibitory effects of anti-telomerase agents as measured by both the TRE and TRAP assays showed a good correlation. Our assay allows precise quantitative comparison of a wide range of human cells from somatic cells to carcinoma cells. TRE assay is suitable for practical use in the assessment of telomerase activity in preclinical and clinical trials of telomerase-based therapies, because of its reproducibility, rapidity and simplicity.     

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).