Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis)

Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.     

Lab and Bench-Scale Pelletization of Torrefied Wood Chips—Process Optimization and Pellet Quality

Combined torrefaction and pelletization is used to increase the fuel value of biomass by increasing its energy density and improving its handling and combustion properties. In the present study, a single-pellet press tool was used to screen for the effects of pellet die temperature, moisture content, additive addition, and the degree of torrefaction on the pelletizing properties and pellet quality, i.e., density, static friction, and pellet strength. Results were compared with pellet production using a bench-scale pelletizer. The results indicate that friction is the key factor when scaling up from single-pellet press to bench-scale pelletizer. Tuning moisture content or increasing the die temperature did not ease the pellet production of torrefied wood chips significantly. The addition of rapeseed oil as a lubricant reduced the static friction by half and stabilized pellet production; however, the pellet quality, strength, and density were negatively affected. The pellets produced from pine wood torrefied at 250 and 280 °C were shorter than pellets produced from untreated wood and their quality did not match conventional wood pellet standards. However, the heating value of the torrefied pellets was higher and the particle size distribution after grinding the pellets was more uniform compared to conventional wood pellets.     

H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.     

Hypercholesterolemia promotes a CD36-dependent and endothelial nitric-oxide synthase-mediated vascular dysfunction

Numerous studies have implicated either the presence or absence of CD36 in the development of hypertension. In addition, hypercholesterolemia is associated with the loss of nitric oxide-induced vasodilation and the subsequent increase in blood pressure. In the current study, we tested the hypothesis that diet-induced hypercholesterolemia promotes the disruption of agonist-stimulated nitric oxide generation and vasodilation in a CD36-dependent manner. To test this, C57BL/6, apoE null, CD36 null, and apoE/CD36 null mice were maintained on chow or high fat diets. In contrast to apoE null mice fed a chow diet, apoE null mice fed a high fat diet did not respond to acetylcholine with a decrease in blood pressure. Caveolae isolated from in vivo vessels did not contain endothelial nitric-oxide synthase and were depleted of cholesterol. Age-matched apoE/CD36 null mice fed a chow or high fat diet responded to acetylcholine with a decrease in blood pressure. The mechanism underlying the vascular dysfunction was reversible because vessels isolated from apoE null high fat-fed mice regained responsiveness to acetylcholine when incubated with plasma obtained from chow-fed mice. Further analysis demonstrated that the plasma low density lipoprotein fraction was responsible for depleting caveolae of cholesterol, removing endothelial nitric-oxide synthase from caveolae, and preventing nitric oxide production. In addition, the pharmacological removal of caveola cholesterol with cyclodextrin mimicked the effects caused by the low density lipoprotein fraction. We conclude that the ablation of CD36 prevented the negative impact of hypercholesterolemia on agonist-stimulated nitric oxide-mediated vasodilation in apoE null mice. These studies provide a direct link between CD36 and the early events that underlie hypercholesterolemia-mediated hypertension and mechanistic linkages between CD36 function, nitric-oxide synthase activation, caveolae integrity, and blood pressure regulation.     

Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.     

Glutathione S transferase M1 andT1 genotypes and susceptibility to smoking related larynx cancer

Susceptibility to smoking related larynx cancer has been suggested to be associated with genetically determined differences in the ability to detoxify carcinogens present in tobacco smoke. The genetic polymorphisms of glutathione S-transferases, involved in the metabolic inactivation of, for example, tobacco derived carcinogens, have been recognized as potential risk modifiers in various environmentally induced malignancies, including larynx cancer. We employed PCR-based methods to determine the distribution of the GSTM 1 and G STT1 null genotypes in 171 larynx cancer patients and 180 controls to examine further their potential role in individual susceptibility to this neoplasm. The GSTM 1 null genotype was found in 49 1 % of the cases and 57 7 % of the controls and the GSTT1 null genotype in 17 5 % of the cases and 21 7 % of the controls, respectively. Larynx cancer risk associated with the lack of GST M 1 (OR = 0 7; 95 % CI: 0 5-1 1) or GSTT1 (OR = 0 8; 95 % CI: 0 5-1 3) was not significantly affected by age, smoking status, or cancer progression. Although this study thus suggests no role for the G STM 1 and GSTT1 gene polymorphisms in individual susceptibility to smoking-related larynx cancer, due to its relatively small sample size more data are required before any definite conclusions can be drawn.     

Characterisation of the cell walls of loquat (Eriobotrya japonica L.) fruit tissues

Loquat fruit (Eriobotrya japonica L. cv. Algor) was dissected to give the following tissue zones: epidermis or epicarp, flesh or mesocarp, integument (a thin layer surrounding the seed cotyledons), seed testa, kernel and hairy receptacle. The alcohol insoluble residues (AIRs) from all these tissues were proved to be free of starch, except loquat kernel which on a fresh weight basis contained about 34% of starch. AIRs were analysed for moisture, ashes, protein, lignin and the component sugars were released by two hydrolytic procedures which helped to distinguish the sugars from non-cellulosic polysaccharides and cellulose. Their major component polysaccharides were inferred to be pectic polysaccharides since all AIRs were very rich in sugars such as uronic acids, arabinose and galactose. Pectic polysaccharides contributed up to 70% of total cell wall polysaccharides in the edible flesh of the loquat fruit. Important differences in the degree of branching, degree of esterification and in the amounts of Ca and Mg associated with pectic polysaccharides were detected among pectic polymers depending on the loquat tissue zone. These compositional and structural differences may be related to the role that these pectic polymers play within the tissues which form the loquat fruit.     

Process for the isolation of preparative quantities of

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997