Cucurbitane triterpenoid oviposition deterrent from Momordica charantia to the leafminer, Liriomyza trifolii

Females of Liriomyza trifolii, a polyphagous leafminer, are often deterred from ovipositing on Momordica charantia leaves. The methanol extract of M. charantia leaves showed strong oviposition deterrent activity against L. trifolii females on the host plant leaf when it was dipped in the methanol extract at a concentration of 1 g of fresh leaf equivalent/ml. Bioguided fractionation of the methanol extract of the leaves of M. charantia led to the isolation of a new cucurbitane triterpenoid, 7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19-al, along with another known compound, momordicine I. Both 7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19-al and momordicine I respectively had significant ovipositing deterrent effect towards the adult females of L. trifolii on host plant leaves treated at concentrations of 3.25 and 33.60 microg/cm2.     

Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells

Sessile bacteria show phenotypical, biochemical, and morphological differences from their planktonic counterparts. Curli, extracellular structures important for biofilm formation, are only produced at temperatures below 30 C in Escherichia coli K-12 strains. In this report, we show that E. coli K-12 can produce curli at 37 C when grown as a biofilm community. The curli-expressing strain formed more biofilms on polyurethane sheets than the curli-deficient strain under growth temperatures of both 25 C and 37 C. Curli are required for the formation of a three-dimensional mature biofilm, with characteristic water channels and pillars of bacteria. Observations by electron microscopy revealed the presence at the surfaces of the curli-deficient mutant in biofilm of flagella and type I pili. A wild-type curli-expressing E. coli strain significantly adhered to several lines of human uroepithelial cells, more so than an isogenic curlideficient strain. The finding that curli are expressed at 37 C in biofilm and enhance bacterial adherence to mammalian host cells suggests an important role for curli in pathogenesis.     

Creation of Rhizopus oryzae lipase having a unique oxyanion hole by combinatorial mutagenesis in the lid domain

Combinatorial libraries of the lid domain of Rhizopus oryzae lipase (ROL; Phe88Xaa, Ala91Xaa, Ile92Xaa) were displayed on the yeast cell surface using yeast cell-surface engineering. Among the 40,000 transformants in which ROL mutants were displayed on the yeast cell surface, ten clones showed clear halos on soybean oil-containing plates. Among these, some clones exhibited high activities toward fatty acid esters of fluorescein and contained non-polar amino acid residues in the mutated positions. Computer modeling of the mutants revealed that hydrophobic interactions between the substrates and amino acid residues in the open form of the lid might be critical for ROL activity. Based on these results, Thr93 and Asp94 were further combinatorially mutated. Among 6,000 transformants, the Thr93Thr, Asp94Ser and Thr93Ser, Asp94Ser transformants exhibited a significant shift in substrate specificity toward a short-chain substrate. Computer modeling of these mutants suggested that a unique oxyanion hole, which is composed of Thr85 Oγ and Ser94 Oγ, was formed and thus the substrate specificity was changed. Therefore, coupling combinatorial mutagenesis with the cell surface display of ROL could lead to the production of a unique ROL mutant.     

Regulation of cell adhesion using a signal-responsive membrane substrate

We have developed a novel cell culture material that regulates cell adhesion by changes in potassium ion concentration. The material is a polyethylene substrate grafted to a copolymer of the thermoresponsive polymer N-isopropylacrylamide (NIPAM) and benzo-18-crown- 6-acrylamide (BCAm), with a pendant crown ether as sensor. The crown ether recognizes potassium ion concentrations and NIPAM conformational changes lead to changes in the hydrophobicity/hydrophilicity balance of the entire polymer at constant cell culture temperatures. Although cells were successfully cultured on the ion recognition material in normal culture medium at 37 degrees C, the cells could be detached from the material surface by adding potassium ions alone, without proteolytic enzymes, because the surface to which the cells were attached altered its surface characteristics to a more hydrophilic state. Therefore, cell layers with intact cell-to-cell junctions and high activities were successfully recovered. Furthermore, by changing the target sensors, this material will be able to control cell adhesion through various cellular signals.     

Phosphorylation of FTDP-17 mutant tau by cyclin-dependent kinase 5 complexed with p35, p25, or p39

One of the major pathological hallmarks of Alzheimer disease is neurofibrillary tangles. Neurofibrillary tangles are bundles of paired helical filaments composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is one of the tau protein kinases that increase paired helical filament epitopes in tau by phosphorylation. Recently, various mutations of tau have been identified in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we investigated the phosphorylation of FTDP-17 mutant tau proteins, K257T, P301L, P301S, and R406W, by Cdk5 complexed with p35, p25, or p39 in vitro and in cultured cells. The extent of phosphorylation by all Cdk5 species was slightly lower in mutant tau than in wild-type tau. Major phosphorylation sites, including Ser202, Ser235, and Ser404, were the same among the wild-type, K257T, P301L, and P301S tau proteins phosphorylated by any Cdk5. On the other hand, R406W tau was less phosphorylated at Ser404 than were the other variants. This was not due to the simple replacement of amino acid Arg406 with Trp close to the phosphorylation site, because Ser404 in a R406W peptide was equally phosphorylated in a wild-type peptide. The decreased phosphorylation of mutant tau by Cdk5s was canceled when tau protein bound to microtubules was phosphorylated. These results indicate that FTDP-17 mutations do not affect the phosphorylatability of tau by Cdk5 complexed with p35, p25, or p39 and may explain part of the discrepancy reported previously between in vivo and in vitro phosphorylation of FTDP-17 tau mutants.     

Effect of diabetes on aortic nitric oxide synthesis in spontaneously hypertensive rats; does captopril modulate this effect?

Nitric oxide (NO) is a potent regulator in the cardiovascular system; it is generated by the nitric oxide synthase (NOS) family of proteins. NO produced in endothelial cells plays a crucial role in vascular functions. The aim of this study was to clarify the effect of diabetes on aortic NO synthesis in a model of genetic hypertension and determine whether captopril modulates this effect. Diabetes was induced in ten weeks old spontaneously hypertensive rats (SHR) by streptozotocin injection. The rats were allocated into 3 groups: control group 1, non-diabetic SHR; group 2, diabetic SHR; group 3, diabetic SHR group receiving captopril at 80 mg/kg in drinking water for 4 weeks. Mean blood pressure (MBP) was measured once a week by tail-cuff method. Aortic NO metabolities (nitrite/nitrate) and endothelial NOS (NOS-3) were assayed by Griess reaction and by immunoblotting and immunohistochemistry, respectively. There was a significant decrease in nitrite/nitrate (NOx) in aortas of diabetic SHR compared with controls. The decrease of aortic NOx in diabetic SHR was accompanied by a decrease in NOS-3 expression. Captopril treatment reduced MBP without affecting either NOx level or NOS-3 expression in aortas of diabetic SHR. We conclude that STZ-induced diabetes decreased NO in aortas of SHR that may reflect endothelial cell dysfunction; captopril administration decreased MBP without affecting NO level in aortas of diabetic SHR which suggest that the blood pressure-lowering effects of captopril were independent of NO.     

Toluene inhalation-induced adrenocortical hypertrophy and endocrinological changes in rat

Rats were exposed to toluene (1,500 ppm for 4 hr per day) for 7 days. The body weight of the rats was significantly lower and the weight of the adrenal gland was significantly higher in the toluene inhalation group compared to the controls. Microscopically, there was no obvious change in the medulla, but hypertrophy of the cortex was observed in the toluene inhalation group. And, the size of adrenocortical cells in treated-rats was also significantly enlarged than the control. Immunohistochemical staining did not show a clear difference in localization of aldosterone-positive cells between the control and inhalation groups. Expansion of the corticosterone-positive area consistent with the cortical hypertrophy was recognized in the inhalation group. Enhancement of 72 kD-heat-shock protein (HSP70)-expression in the toluene inhalation group was not observed. Neither stress nor damage to cortical cells due directly to toluene exposure was observed in the cortex. Also, there was no obvious difference in the anti-proliferating cell nucleus antigen (PCNA)-immunostaining between control and inhalation groups. Thus, it is suspected that cortical hypertrophy was the result of cell enlargement due to the stimulation of the cortical cells. Corticotropin-releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) was increased in the inhalation group. Concentration of plasma ACTH was elevated significantly by toluene exposure. The amounts of mRNA of adrenocortical steroid metabolism gene, cytochrome side-chain cleavage (P450scc), was also increased by toluene inhalation. Toluene exposure might induce adrenocortical hypertrophy via the hypothalamus-pituitary-adrenal gland (HPA) axis.     

Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins

Galectin-9 and galectin-8, members of beta-galactoside-binding animal lectin family, are promising agents for the treatment of immune-related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin-9 and galectin-8.