Growth rates of Eucheuma denticulatum (Burman) Collins et Harvey and Kappaphycus striatum (Schmitz) Doty under different conditions in warm waters of Southern Japan

Green and brown strains of Eucheuma denticulatum and new cultivated strain Kappaphycus striatum were collected from commercial cultivation ground in Philippines. Daily growth rates (DGR) of the materials were measured under different temperatures and photon fluence in laboratory controlled conditions and at different depths in the sea using the floating raft culture method in Uranochi Inlet, Tosa Bay, Southern Japan. The highest DGR of E. denticulatum (brown strain) (2.76% ±0.64) and K. striatum (green strain) (4.5% ±1.51) were recorded at 25°C. K. striatum (both strains) had the highest DGR and grew over a wide range of temperatures. Observation on the effect of photon fluence showed that K. striatum (brown strain) grew well (DGR = 5.16%±1.3) at 145 μmol photon m^-2 s^-1.In the floating raft cultivation, K. striatum (green strain) had a DGR of3.1% ± 0.93 higher than E. denticulatum (brown strain)(2.73% ± 0.43). This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytochrome c dissolved in 1-allyl-3-methylimidazolium chloride type ionic liquid undergoes a quasi-reversible redox reaction up to 140°C

Solubility of cytochrome c, a typical heme protein, in a various ionic liquids has been analyzed. The solubility has been discussed with polarity parameters of the ionic liquids. Both hydrogen bond basicity and dipolarity/polarizability of the ionic liquids were confirmed to be influential factors to control the solubilization of cytochrome c. Polar ionic liquids such as 1-butyl-3-methylimidazolium chloride and 1-allyl-3-methylimidazolium chloride solubilized cytochrome c at 80°C, and the dissolved cytochrome c was found to keep its redox activity in these ionic liquids. The redox response of the dissolved cytochrome c was detected in 1-allyl-3-methylimidazolium chloride up to 140°C.     

Prolonged, NK cell-mediated antitumor effects of suicide gene therapy combined with monocyte chemoattractant protein-1 against hepatocellular carcinoma

Tumor recurrence rates remain high after curative treatments for hepatocellular carcinoma (HCC). Immunomodulatory agents, including chemokines, are believed to enhance the antitumor effects of tumor cell apoptosis induced by suicide gene therapy. We therefore evaluated the immunomodulatory effects of a bicistronic recombinant adenovirus vector (rAd) expressing both HSV thymidine kinase and MCP-1 on HCC cells. Using an athymic nude mouse model (BALB/c-nu/nu), primary s.c. tumors (HuH7; human HCC cells) were completely eradicated by rAd followed by treatment with ganciclovir. The same animals were subsequently rechallenged with HCC cells, tumor development was monitored, and the recruitment or activation of NK cells was analyzed immunohistochemically or by measuring IFN-gamma mRNA expression. Tumor growth was markedly suppressed as compared with that in mice treated with a rAd expressing the HSV thymidine kinase gene alone (p < 0.001). Suppression of tumor growth was associated with the elevation of serum IL-12 and IL-18. During suppression, NK cells were recruited exclusively, and Th1 cytokine gene expression was enhanced in tumor tissues. The antitumor activity, however, was abolished either when the NK cells were inactivated with anti-asialo GM1 Ab or when anti-IL-12 and anti-IL-18 Abs were administered. These results indicate that suicide gene therapy, together with delivery of MCP-1, eradicates HCC cells and exerts prolonged NK cell-mediated antitumor effects in a model of HCC, suggesting a plausible strategy to prevent tumor recurrence.     

The residual structure of acid-denatured β2-microglobulin is relevant to an ordered fibril morphology

β₂-Microglobulin (β2m) forms amyloid fibrils in vitro under acidic conditions. Under these conditions, the residual structure of acid-denatured β2m is relevant to seeding and fibril extension processes. Disulfide (SS) bond-oxidized β2m has been shown to form rigid, ordered fibrils, whereas SS bond-reduced β2m forms curvy, less-ordered fibrils. These findings suggest that the presence of an SS bond affects the residual structure of the monomer, which subsequently influences the fibril morphology. To clarify this process, we herein performed NMR experiments. The results obtained revealed that oxidized β2m contained a residual structure throughout the molecule, including the N- and C-termini, whereas the residual structure of the reduced form was localized and other regions had a random coil structure. The range of the residual structure in the oxidized form was wider than that of the fibril core. These results indicate that acid-denatured β2m has variable conformations. Most conformations in the ensemble cannot participate in fibril formation because their core residues are hidden by residual structures. However, when hydrophobic residues are exposed, polypeptides competently form an ordered fibril. This conformational selection phase may be needed for the ordered assembly of amyloid fibrils.     

Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.

An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.     

Sudan III dye strongly induces CYP1A1 mRNA expression in HepG2 cells

Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 μM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III--treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III.     

Effects of dietary calcium restriction and acute exercise on the antioxidant enzyme system and oxidative stress in rat diaphragm

Calcium deficiency is considered to increase intracellular calcium level; thus the aim of the current study was to elucidate whether dietary calcium restriction enhanced exercise-induced oxidative stress in rat diaphragm. Twenty male Wistar rats were randomly assigned to either a control group or a group subjected to 1 mo of calcium restriction. In addition, each group was subsequently subdivided into rested or acutely exercised group. Dietary calcium restriction significantly (P < 0.05) upregulated the activities of manganese-superoxide dismutase (Mn-SOD), copper-zinc-superoxide dismutase (Cu-Zn-SOD), and glutathione peroxidase (Gpx) but not catalase. Acute exercise, in addition to calcium restriction, decreased both SOD isoenzymes in the diaphragm of calcium-restricted rats (P < 0.05). On the other hand, calcium restriction resulted in increased Gpx mRNA expression (P < 0.05). In control rats, acute exercise significantly (P < 0.05) increased the expressions of both SOD mRNAs, whereas in the calcium-restricted rats, it increased that of Mn-SOD mRNA (P < 0.05) but decreased that of Gpx mRNA (P < 0.05). Furthermore, reactive carbonyl derivative, a marker of protein oxidation, was significantly greater in the calcium-restricted rats than in the control rats after acute exercise (P < 0.05). The results suggest that antioxidant enzymes in rat diaphragm were upregulated in response to an increased oxidative stress by dietary calcium restriction but that upregulation is not enough to cope with exercise-induced further increase of oxidative stress.     

Epimorphin acts extracellularly to promote cell sorting and aggregation during the condensation of vertebral cartilage

Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.     

Long untranslated regions of the measles virus M and F genes control virus replication and cytopathogenicity

Measles is still a major cause of mortality mainly in developing countries. The causative agent, measles virus (MeV), is an enveloped virus having a nonsegmented negative-sense RNA genome, and belongs to the genus Morbillivirus of the family Paramyxoviridae. One feature of the moribillivirus genomes is that the M and F genes have long untranslated regions (UTRs). The M and F mRNAs of MeV have 426-nucleotide-long 3' and 583-nucleotide-long 5' UTRs, respectively. Though these long UTRs occupy as much as approximately 6.4% of the virus genome, their function remains unknown. To elucidate the role of the long UTRs in the context of virus infection, we used the reverse genetics based on the virulent strain of MeV, and generated a series of recombinant viruses having alterations or deletions in the long UTRs. Our results showed that these long UTRs per se were not essential for MeV replication, but that they regulated MeV replication and cytopathogenicity by modulating the productions of the M and F proteins. The long 3' UTR of the M mRNA was shown to have the ability to increase the M protein production, promoting virus replication. On the other hand, the long 5' UTR of the F mRNA was found to possess the capacity to decrease the F protein production, inhibiting virus replication and yet greatly reducing cytopathogenicity. We speculate that the reduction in cytopathogenicity may be advantageous for MeV fitness and survival in nature.