Demonstration of a Heat-Stable 120-Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group-Common Antigen

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as λRj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and λgt11 lacZ gene amplified the λRj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.     

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran

A highly sensitive ATP bioluminescence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli.     

Changes in plasma alpha and gamma tocopherol levels before and after long-term local hyperthermia in cancer patients

Local hyperthermia is one of the heat therapies for cancer patients. The effect of this therapy is recognized to affect the immune function. On the other hand, researchers have recently suggested that vitamin E has not only antioxidant but also other functions including the immune function. However, the association between local hyperthermia therapy and vitamin E level is not yet well understood. Comparing plasma alpha and gamma tocopherol levels before and after the therapy, the basal levels of both tocopherols in the cancer patients did not significantly differ from those in healthy subjects. However, the interindividual difference in the basal levels was very wide in the cancer patients. After long-term local hyperthermia (more than 70 days), the levels of both tocopherols were significantly higher than the basal levels. This result suggests that long-term local hyperthermia therapy influences plasma tocopherol level in cancer patients; thus, an increase in vitamin E level may play an important role in the therapy of cancer patients.     

Relationship between chain collapse and secondary structure formation in a partially folded protein

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil‐globule transition)? To answer this question, we measured small‐angle X‐ray scattering for a series of β‐lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil‐globule transitions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 651–658, 2014.     

sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB—5-fluoroorotic acid (5FOA)—pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura⁻) and hence resistant to 5FOA (5FOA^r). Although Ura⁺ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA^r to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA^r selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA^r colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

Singular behavior of slow dynamics of single excitable cells

In various kinds of cultured cells, it has been reported that the membrane potential exhibits fluctuations with long-term correlations, although the underlying mechanism remains to be elucidated. A cardiac muscle cell culture serves as an excellent experimental system to investigate this phenomenon because timings of excitations can be determined over an extended time period in a noninvasive manner through visualization of contractions, although the properties of beat-timing fluctuations of cardiac muscle cells at the single-cell level remains to be fully clarified. In this article, we report on our investigation of spontaneous contractions of cultured rat cardiac muscle cells at the single-cell level. It was found that single cells exhibit several typical temporal patterns of contractions and spontaneous transitions among them. Detrended fluctuation analysis on the time series of interbeat intervals revealed the presence of 1/f^β noise at sufficiently large timescales. Furthermore, multifractality was also found in the time series of interbeat intervals. These experimental trends were successfully explained using a simple mathematical model, incorporating correlated noise into ionic currents. From these findings, it was established that singular fluctuations accompanying 1/f^β noise and multifractality are intrinsic properties of single cardiac muscle cells.     

Natural killer cell is a major producer of interferon γ that is critical for the IL-12-induced anti-tumor effect in mice

Although the anti-tumor effect of IL-12 is mediated mostly by IFNγ, which cell types most efficiently produce IFNγ and therefore initiate or promote the anti-tumor effect of IL-12 has not been clearly determined. In the present study, we demonstrated hydrodynamic injection of the IL-12 gene led to prolonged IFNγ production, NK-cell activation and complete inhibition of liver metastasis of CT-26 colon cancer cells in wild-type mice, but not in IFNγ knockout mice. NK cells expressed higher levels of STAT4 and upon IL-12 administration displayed stronger STAT4 phosphorylation and IFNγ production than non-NK cells. Adoptive transfer of wild-type NK cells into IFNγ knockout mice restored IL-12-induced IFNγ production, NK-cell activation and anti-tumor effect, whereas transfer of the same number of wild-type non-NK cells did not. In conclusion, NK cells are predominant producers of IFNγ that is critical for IL-12 anti-tumor therapy.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.