A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.     

Mycotoxin Production by Fusarium proliferatum isolates from rice with Fusarium sheath rot disease

Twenty samples of unpolished (rough) rice collected in Arkansas and Texas during the 1995 harvesting season from fields exhibiting Fusarium sheath rot disease or panicle blight were previously shown to include 8 samples positive for fumonisin B₁(FB₁) in the range 2.2–5.2 ppm, and moniliformin (MON), but no beauvericin (BEA), deoxynivalenol, its derivatives or zearalenone were detected. Fifteen cultures of F. proliferatum were established from the 20 rough rice samples. Single spore isolates of each culture were grown on rice and tested for the production of fumonisins (FB₁, FB₂, FB₃, etc.), MON and BEA. All 15 isolates produced FB₁, FB₂, MON and BEA in culture on rice. No deoxynivalenol, its derivatives orzearalenone were detected. Seven cultures produced FB₁ at >50ppm (range 80–230 ppm), with therest producing FB₁ in the range 14–43 ppm.FB₂ was produced in the range 5–47 ppm, and those cultures which produced the most FB₁ also produced the most FB₂. Of the 15 cultures producing MON, 11 produced it at >100 ppm in the range 188–6018 ppm, with the rest producing in the range 7–64 ppm. BEA was produced in the range 109–1350 ppm. Other derivatives of fumonisins, including FA₁, FA₂ and partially hydrolyzed FB₁, as well asseveral unknown metabolites including a compound with MW 414, were identified in culture extracts by continuous flow fast atom bombardment with ion spraymass spectrometry (CF/FAB/MS). Further study is needed to identify the factors that control production of FB₁, MON and BEA by F.proliferatu in culture and in field samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis and anticonvulsant activity of new 2-substituted-5- [2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles and 1,2,4-triazoles

A series of new 2-substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles has been synthesized and screened for their anticonvulsant activities. Compound 3 shows considerable anticonvulsant activity both in PTZ and MES models. It seems that this effect is mediated by benzodiazepine receptors and other unknown mechanism, respectively.     

(E)- and (Z)-1,2,4-triazolylchromanone oxime ethers as conformationally constrained antifungals

A series of 1,2,4-triazolylchromanone oxime ethers were synthesized and tested for in vitro antifungal activity. Many of these derivatives exhibit high activity against Candida albicans, Saccharomyces cerevisiae, Aspergillus niger and Microsporum gypseum.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.     

Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin

Theactin-binding proteins dystrophin and -actinin are members of afamily of actin-binding proteins that may link the cytoskeleton tomembrane proteins such as ion channels. Previous work demonstrated thatthe activity of Ca²⁺ channels can be regulated by agentsthat disrupt or stabilize the cytoskeleton. In the present study, weemployed immunohistochemical and electrophysiological techniques toinvestigate the potential regulation of cardiac L-type Ca²⁺channel activity by dystrophin and -actinin in cardiac myocytes andin heterologous cells. Both actin-binding proteins were found tocolocalize with the Ca²⁺ channel in mouse cardiac myocytesand to modulate channel function. Inactivation of the Ca²⁺channel in cardiac myocytes from mice lacking dystrophin(mdx mice) was reduced compared with that in wild-typemyocytes, voltage dependence of activation was shifted by 5 mV to morepositive potentials, and stimulation by the -adrenergic pathway andthe dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca²⁺ channel with muscle,but not nonmuscle, forms of -actinin was also found to reduceinactivation. As might be predicted from a reduction ofCa²⁺ channel inactivation, a prolonging of the mouseelectrocardiogram QT was observed in mdx mice. These resultssuggest a combined role for dystrophin and -actinin in regulatingthe activity of the cardiac L-type Ca²⁺ channel and apotential mechanism for cardiac dysfunction in Duchenne and Beckermuscular dystrophies.

     

Phytotoxicity and ultrastructural effects of gymnopusin from the orchid Maxillaria densa on duckweed (Lemna pausicostata) frond and root tissues

The effect of life cycle on the distributions of C(25) and C(30) highly branched isoprenoid (HBI) alkene lipids has been investigated for the marine diatom Rhizosolenia setigera. The concentrations of the C(30) compounds are largely independent of the cell volume, though the ratios of the individual isomers possessing five and six double bonds show a dependence on the position of the cell during its life cycle, especially during auxosporulation. In contrast to the C(30) pseudo-homologues, the C(25) isomers are not always detected in cultures of R. setigera. The biosynthesis of the C(25) HBIs would appear to result from the onset of auxosporulation, with further changes to their distributions taking place after this phase, including the formation of more unsaturated isomers. The results of this investigation may be used in part to explain the large variations in these lipids reported previously.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Microbial hydroxylation of progesterone with Acremonium strictum

Microbial hydroxylation of progesterone occurred in the culture of Acremonium strictum PTCC 5282 to produce two hydroxylated pregnene-like steroids. The metabolites were purified and characterized using spectroscopic methods and identified as 15alpha-hydroxyprogesterone and 15alpha-hydroxydeoxycorticosterone.