Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

Saffold Virus Type 3 (SAFV-3) Persists in HeLa Cells

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler’s murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

A tightly regulated expression system for <Emphasis Type="Italic">E. coli</Emphasis> using supersaturated silicic acid

Objective

To develop a new expression system regulated by a ferric uptake regulator in which silicic acid is used as an inducer.

Results

Fur box (binding site for Fur) was substituted for the lac operator to regulate the expression of GFP with the lac promoter. Since the addition of supersaturated silicic acid invokes iron deficiency, supersaturated silicic acids were used as an inducer. GFP expression was dependent on silica concentration, and the expression level without silica was negligible. Basal expression level of this lac-Fur system was extremely low and, hence, lytic enzyme gene E from bacteriophage ?X174 could be retained in this system. Furthermore, the expression of genes of interest was spontaneously initiated as the cell density increased and the costs of the inducer are considerably less than IPTG.

Conclusion

The combination of lac promoter and Ferric uptake repressor allowed the protein expression by supersaturated silicic acid as an inducer in an easy and cost-effective way.

     

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.     

Increase in respiratory cost at high growth temperature is attributed to high protein turnover cost in Petunia x hybrida petals

It is widely believed that turnover of nitrogenous (N) compounds (especially proteins) incurs a high respiratory cost. Thus, if protein turnover costs change with temperature, this would influence the dependence of respiration rate on growth temperature. Here, we examined the extent to which protein turnover cost explained differences in N-utilization costs (nitrate uptake/reduction, ammonium assimilation, amino acid and protein syntheses, protein turnover and amino acid export) and in respiration rate with changes in growth temperature. By measurements and literature data, we evaluated each N-utilization cost in Petunia x hybrida petals grown at 20, 25 or 35 degrees C throughout their whole lifespans. Protein turnover cost accounted for 73% of the integrated N-utilization cost on a whole-petal basis at 35 degrees C. The difference in this cost on a dry weight basis between 25 and 35 degrees C accounted for 75% of the difference in N-utilization cost and 45% of the difference in respiratory cost. The cost of nitrate uptake/reduction was high at low growth temperatures. We concluded that respiratory cost in petals was strongly influenced by protein turnover and nitrate uptake/reduction, and on the shoot basis, C investment in biomass was highest at 25 degrees C.