Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Subcellular Localization of Sorbitol-6-phosphate Dehydrogenase in Protoplast from Apple Cotyledons

The subcellular localization of sorbitol-6-phosphate (S6P) dehydrogenasein protoplasts of apple cotyledons was examined by differentialcentrifugation and linear sucrose density gradient centrifugation(30–60%, w/w). The distribution of S6P dehydrogenase activitywas 55% in the 500 x g pellet of the homogenate and 35% in thesupernatant of 105,000 x g. When the x g pellet was recentrifugedin a linear sucrose density gradient, one major peak of activitywas found at a density of 1.23. This peak coincided with themajor peak of chlorophyll and NADP⁺-triose-P dehydrogenase activity.When the 500 x g pellet was sonicated, the major peak of S6Pdehydrogenase activity shifted to a lighter density (d=1.18).The shifted peak also coincided with the peak of chlorophyll.The enzyme detected in the major peak of chlorophyll (d=1.23)was partially solubilized by sonic or detergent treatment, butnot by hypotonic solution. The results supported the localizationof S6P dehydrogenase in chloroplasts, and presumably their associationwith thylakoid membranes. Part of the enzyme was assumed tobe naturally present in the cytosol, too. (Received November 4, 1980; Accepted January 21, 1981)     

Molecular identification of the causative agent of human strongyloidiasis acquired in Tanzania: Dispersal and diversity of Strongyloides spp. and their hosts

In order to identify the causative agent of imported strongyloidiasis found in a Japanese mammalogist, who participated in a field survey in Tanzania, the hyper-variable region IV (HVR-IV) of 18S ribosomal DNA and partial mitochondrial cytochrome c-oxidase subunit 1 gene (cox1) were analyzed and compared with Strongyloides fuelleborni collected from apes and monkeys of Africa and Japan, and S. stercoralis from humans, apes and dogs. The HVR-IV and cox1 of the patient's worms were identical to or only slightly differed from those of worms parasitic in Tanzanian chimpanzees and yellow baboons, demonstrating that the patient acquired the infection during her field survey in Tanzania. Phylogenetic analysis with the maximum-likelihood method largely divided isolates of S. fuelleborni into three groups, which corresponded to geographical localities but not to host species. Meanwhile, isolates of S. stercoralis were grouped by the phylogenetic analysis into dog-parasitic and primate-parasitic clades, and not to geographical regions. It is surmised that subspeciation has occurred in S. fuelleborni during the dispersal of primates in Africa and Asia, while worldwide dispersal of S. stercoralis seems to have occurred more recently by migration and the activities of modern humans.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Innate immune response to viral infection

In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.     

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)- 2 and (−)- 2 , respectively, from 310 to 210 nm. Furthermore, (−)- 2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.     

Farnesyltransferase inhibitor improved survival following endotoxin challenge in mice

Endotoxemia plays an important role in the pathogenesis of sepsis and is accompanied by dysregulated apoptosis of immune and non-immune cells. Treatment with statins reduces mortality in rodent models of sepsis and endotoxemia. Inhibition of protein isoprenylation, including farnesylation, has been proposed as a mechanism to mediate the lipid-lowering-independent effects of statins. Nonetheless, the effects of the inhibition of isoprenylation have not yet been studied. To investigate the role of farnesylation, we evaluated the effects of farnesyltransferase inhibitor and statin on survival following lipopolysaccharide (LPS) challenge in mice. Both simvastatin (2 mg/kg BW) and FTI-277 (20 mg/kg BW) treatment improved survival by twofold after LPS injection, as compared with vehicle alone (p < 0.01). LPS-induced cleavage (activation) of caspase-3, an indicator of apoptotic change, and increased protein expression of proapoptotic molecules, Bax and Bim, and activation of c-Jun NH₂-terminal kinase (JNK/SAPK) in the liver and spleen were attenuated by both simvastatin and FTI-277. These results demonstrate that farnesyltransferase inhibitor as well as statin significantly reduced LPS-induced mortality in mice. Our findings also suggest that inhibition of protein farnesylation may contribute to the lipid-lowering-independent protective effects of statins in endotoxemia, and that protein farnesylation may play a role in LPS-induced stress response, including JNK/SAPK activation, and apoptotic change. Our data argue that farnesyltransferase may be a potential molecular target for treating patients with endotoxemia.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.