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.     

Development of seaweed communities on suspended substrata with three slope angles

Observations on the development of seaweed communities on concretepanels suspended at three different slope angles – 0^° (horizontal),45^° (inclined) and 90^° (vertical) – were carried out atUranouchi Inlet, Tosa Bay, southern Japan. Each panel had two opposite20 × 20 cm areas, which were affixed with six pieces of 6 × 9 cmacrylic plate on which algal communities were allowed to colonize. The panelswere suspended 1 m below the sea surface from a floating platform fora period of 6 weeks. The experiments were repeated nine times fromAugust 1996 to October 1997. Young thalli could already be recognized on the panels after 3–4 weeks. Thetype of algal community developing on a panel varied with the slope angleas well as the period of suspension. These were classified into the threedominant green algal genera: Ulva, Enteromorpha and Cladophora. The cover and biomass of particular species were clearlyinfluenced by the slope of the substratum.     

Construction of an expression system for aqualysin I in Escherichia coli that gives a markedly improved yield of the enzyme protein

An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Delta) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 degrees C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 degrees C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNDeltaC105. By analyzing the product of the pMAQ-c2Delta-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.     

Soluble MICA and a MICA Variation as Possible Prognostic Biomarkers for HBV-Induced Hepatocellular Carcinoma

MHC class I polypeptide-related chain A (MICA) molecule is induced in response to viral infection and various types of stress. We recently reported that a single nucleotide polymorphism (SNP) rs2596542 located in the MICA promoter region was significantly associated with the risk for hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) and also with serum levels of soluble MICA (sMICA). In this study, we focused on the possible involvement of MICA in liver carcinogenesis related to hepatitis B virus (HBV) infection and examined correlation between the MICA polymorphism and the serum sMICA levels in HBV-induced HCC patients. The genetic association analysis revealed a nominal association with an SNP rs2596542; a G allele was considered to increase the risk of HBV-induced HCC (P = 0.029 with odds ratio of 1.19). We also found a significant elevation of sMICA in HBV-induced HCC cases. Moreover, a G allele of SNP rs2596542 was significantly associated with increased sMICA levels (P = 0.009). Interestingly, HCC patients with the high serum level of sMICA (>5 pg/ml) exhibited poorer prognosis than those with the low serum level of sMICA (≤5 pg/ml) (P = 0.008). Thus, our results highlight the importance of MICA genetic variations and the significance of sMICA as a predictive biomarker for HBV-induced HCC.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

A one-step enzyme immunoassay for human manganese superoxide dismutase with monoclonal antibodies

A one-step enzyme immunoassay for the determination of manganese superoxide dismutase in serum has been developed with two kinds of monoclonal antibodies. Proposed method had high sensitivity (assay range, 0.4-200 ng/ml), good recovery (recovery percentage, 102.9-106.2%) and reproducibility (intraassay, C.V. = 1.87-3.66%; interassay, C.V. = 3.03-10.4%). From these results, it is possible to apply this method to routine clinical analysis and biochemical research with various purposes.     

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Serum level of advanced glycation end-products (AGEs) is an independent determinant of plasminogen activator inhibitor-1 (PAI-1) in nondiabetic general population.

Glucose can react nonenzymatically with amino groups of proteins to form senescent macroprotein derivatives termed advanced glycation end-products (AGEs). Recently, AGEs have been shown to play an important role in atherosclerosis even in nondiabetic subjects. However, the molecular mechanism underlying this is not fully understood. We have now investigated whether serum AGE level was an independent determinant of plasminogen activator inhibitor-1 (PAI-1), a major physiological inhibitor of fibrinolysis, in nondiabetic general population. One-hundred and eighty-six nondiabetic Japanese subjects underwent a complete history and physical examination, determination of blood chemistries, PAI-1, and AGEs. Uni- and multivariate analyses were applied for the determinants of PAI-1 levels. The average PAI-1 levels were 29.7+/-23.8 ng/ml in males and 21.8+/-17.1 ng/ml in females, respectively. Univariate regression analysis showed that PAI-1 levels were associated with age (inversely, p=0.003), male (p=0.003), body mass index (BMI) (p<0.001), HDL-cholesterol (inversely, p<0.001), triglycerides (p<0.001), fasting plasma glucose (p<0.001), insulin (p<0.001), uric acids (p<0.001), AGEs (p=0.037), and alcohol intake (p<0.001). By the use of multiple regression analyses, BMI (p<0.001), male (p=0.003), fasting plasma glucose (p=0.005), age (inversely, p=0.017), and AGEs (p=0.034) remained significant. The present study is the first demonstration that serum AGE level was one of the independent determinants of PAI-1 in nondiabetic general population. The AGE-associated thrombogenic abnormality may be involved in atherogenesis in nondiabetic subjects.     

Type F scavenger receptor SREC-I interacts with advillin, a member of the gelsolin/villin family, and induces neurite-like outgrowth

The scavenger receptor expressed by endothelial cells (SREC) was isolated from a human endothelial cell line and consists of two isoforms named SREC-I and -II. Both isoforms have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in the extracellular domains and have unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions. The extracellular domain of SREC-I binds modified low density lipoprotein and mediates a homophilic SREC-I/SREC-I or heterophilic SREC-I/SREC-II trans-interaction. However, the significance of large Ser/Pro-rich cytoplasmic domains of SRECs is not clear. Here, we found that when SREC-I was overexpressed in murine fibroblastic L cells, neurite-like outgrowth was induced, indicating that the receptor can lead to changes in cell morphology. The SREC-I-mediated morphological change required the cytoplasmic domain of the protein, and we identified advillin, a member of the gelsolin/villin family of actin regulatory proteins, as a protein binding to this domain. Reduction of advillin expression in L cells by RNAi led to the absence of the described SREC-I-induced morphological changes, indicating that advillin is a prerequisite for the change. Finally, we demonstrated that SREC-I and advillin were co-expressed and interacted with each other in dorsal root ganglion neurons during embryonic development and that overexpression of both SREC-I and advillin in cultured Neuro-2a cells induced long process formation. These results suggest that the interaction of SREC-I and advillin are involved in the development of dorsal root ganglion neurons by inducing the described morphological changes.