Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Inhibition of constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis

Constitutive activation of the NF-kappaB has been documented to be involved in the pathogenesis of many human malignancies, including hemopoietic neoplasms. In this study, we examined the status of NF-kappaB in two non-Hodgkin's lymphoma cell lines derived from mantle cell lymphoma (MCL) samples and in patient MCL biopsy specimens by EMSA and confocal microscopic analysis. We observed that NF-kappaB is constitutively activated in both the MCL cell lines and in the MCL patient biopsy cells. Since NF-kappaB has been shown to play an important role in a variety of cellular processes, including cell cycle regulation and apoptosis, targeting the NF-kappaB pathways for therapy may represent a rational approach in this malignancy. In the MCL cell lines, inhibition of constitutive NF-kappaB by the proteasome inhibitor PS-341 or a specific pIkappaBalpha inhibitor, BAY 11-7082, led to cell cycle arrest in G(1) and rapid induction of apoptosis. Apoptosis was associated with the down-regulation of bcl-2 family members bcl-x(L) and bfl/A1, and the activation of caspase 3, that mediates bcl-2 cleavage, resulting in the release of cytochrome c from the mitochondria. PS-341or BAY 11-induced G(1) cell cycle arrest was associated with the inhibition of cyclin D1 expression, a molecular genetic marker of MCL. These studies suggest that constitutive NF-kappaB expression plays a key role in the growth and survival of MCL cells, and that PS-341 and BAY 11 may be useful therapeutic agents for MCL, a lymphoma that is refractory to most current chemotherapy regimens.     

Expression of a heterologous S-adenosylmethionine decarboxylase cDNA in plants demonstrates that changes in S-adenosyl-L-methionine decarboxylase activity determine levels of the higher polyamines spermidine and spermine

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.     

5 alpha-reductase-catalyzed conversion of testosterone to dihydrotestosterone is increased in prostatic adenocarcinoma cells: suppression by 15-lipoxygenase metabolites of gamma-linolenic and eicosapentaenoic acids

Although the androgens, testosterone (T) and its highly active metabolite dihydrotestosterone (DHT) play a role in the development and progression of prostate cancer, the mechanism(s) are unclear. Furthermore, 5 alpha-reductase which catalyze the conversion of T to DHT, has been a target of manipulation in the treatment of prostatic cancer, hence synthetic 5 alpha-reductase activity inhibitors have shown therapeutic promise. To demonstrate that nutrients derived from dietary sources can exert similar therapeutic promise, this study was designed using benign hyperplastic cells (BHC) and malignant tumorigenic cells (MTC) derived from Lobund-Wistar (L-W) rat model of prostatic adenocarcinoma to test the effects of gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and their 15-lipoxygenase metabolites on cellular 5 alpha-reductase activity. Our data revealed: (i) that incubation of MTC with [3H]-T resulted in marked conversion to [3H]-DHT when compared to similar incubation with BHC; (ii) that DHT-enhanced activity of 5 alpha-reductase was inhibited 80% by 15S-hydroxyeicosatrienoic acid, the 15-lipoxygenase metabolite of GLA, when compared to 55% by 15S-hydroxyeicosapentaenoic acid, the 15-lipoxygenase metabolite of EPA; and (iii) that their precursor fatty acids, respectively, exerted moderate inhibition. Taken together, the study underscores the biological importance of 15-lipoxygenase metabolites of polyunsaturated fatty acids (PUFAs) in androgen metabolism.     

Protein micelles from lipoxygenase 3

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.     

Xanthones from the bark of Garcinia merguensis

The bark of Garcinia merguensis yielded 10 xanthones, merguenone, 1,5-dihydroxy-6'-methyl-6'-(4-methyl-3-pentenyl)-pyrano(2',3':3,2)-xanthone, subelliptenone H, 8-deoxygartanin, rheediaxanthone A, morusignin G, 6-deoxyjacareubin, 1,3,5-trihydroxy-4,8-di(3-methylbut-2-enyl)-xanthone, rheediachromenoxanthone and 6-deoxyisojacareubin. The structure of merguenone was determined using spectroscopic techniques, mainly 1D and 2D NMR spectroscopy.     

Identification and mapping of the QTL for aluminum tolerance introgressed from the new source, <Emphasis Type="SmallCaps">ORYZA RUFIPOGON</Emphasis> Griff., into indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

This study was conducted to identify and map the quantitative trait locus (QTL) controlling Al tolerance in rice using molecular markers. A population of 171 F(6) recombinant inbred lines (RILs) derived from the cross of Oryza sativa (IR64), the Al susceptible parent, and Oryza rufipogon, the Al tolerant parent, was evaluated for Al tolerance using a nutrient solution with and without 40 ppm of active Al(+3). A genetic map, consisting of 151 molecular markers covering 1,755 cM with an average distance of 11.6 cM between loci, was constructed. Nine QTLs were dentified including one for root length under non-stress conditions (CRL), three for root length under Al stress (SRL) and five for relative root length (RRL). O. rufipogon contributed favorable alleles for each of the five QTLs for RRL, which is a primary parameter for Al tolerance, and individually they explained 9.0-24.9% of the phenotypic variation. Epistatic analysis revealed that CRL was conditioned by an epistatic effect, whereas SRL and RRL were controlled by additive effects. Comparative genetic analysis showed that QTLs for RRL, which mapped on chromosomes 1 and 9, appear to be consistent among different rice populations. Interestingly, a major QTL for RRL, which explained 24.9% of the phenotypic variation, was found on chromosome 3 of rice, which is conserved across cereal species. These results indicate the possibilities to use marker-assisted selection and pyramiding QTLs for enhancing Al tolerance in rice. Positional cloning of such QTLs introgressed from O. rufipogon will provide a better understanding of the Al tolerance mechanism in rice and the evolutionary genetics of plant adaptation to acid-soil conditions across cereal species.     

High efficiency synthesis of a bioconjugatable near-infrared fluorochrome

Near-infrared (NIR) fluorochromes have become important reporter molecules for many biomedical applications, including FACS sorting, confocal microscopy, and more recently in vivo imaging. While the structures of several stable 800 nm fluorochromes have been published, their synthesis is often complex and there are difficulties in rapidly purifying these compounds in large quantities. Here we report on the synthesis of NIR820, ex/em = 790/820, with excellent physicochemical properties. Importantly, NIR820 is conveniently synthesized in a three-step reaction and can be purified by flash column chromatography rather than by HPLC. NIR820 is chemically stable and can be directly coupled to peptides during the solid-phase synthesis. In addition, NIR820 is also suitable for conjugation to proteins and other affinity molecules in aqueous buffer.