Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.     

A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.     

Assessment of human adenovirus removal by qPCR in an advanced water reclamation plant in Georgia,USA

Aims

To assess human adenoviruses (HAdVs) removal in an advanced wastewater treatment facility and compare two parallel tertiary treatment methods for the removal of HAdVs.

Methods and Results

Tangential flow ultrafiltration was used to concentrate the water samples, and HAdVs were precipitated by polyethylene glycol. HAdVs were detected only by TaqMan real‐time PCR, and HAdV genotype was determined by DNA sequence. HAdVs were detected in 100% of primary clarification influent, secondary clarification effluent and granular media (GM) filtration effluent samples but only in 31·2% of membrane filtration (MF) effluent and 41·7% of final effluent (FE) samples, respectively. The average HAdVs loads were significantly reduced along the treatments but HAdVs were still present in FE. Comparison of two parallel treatments (GM vs MF) showed that MF was technically superior to GM for the removal of HAdVs.

Conclusions

These findings indicate that adenoviruses are not completely removed by treatment processes. MF is a better treatment for removal of adenoviruses than GM filtration. Because only qPCR was used, the results only indicate the removal of adenovirus DNA and not the infectivity of viruses.

Significance and Impact of the Study

Presence of HAdVs in FE by qPCR suggests a potential public health risk from exposure to the treated wastewater and using the FE for recreational or water reuse purposes should be cautious.     

Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Separation of the Saccharomyces cerevisiae Paf1 complex from RNA polymerase II results in changes in its subnuclear localization

The yeast Paf1 complex (Paf1C), composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, associates with RNA polymerase II (Pol II) at promoters and in the actively transcribed portions of mRNA genes. Loss of Paf1 results in severe phenotypes and significantly reduced levels of the other Paf1C components. In contrast, loss of Rtf1 causes relatively subtle phenotypic changes and no reduction in the other Paf1C factors but disrupts the association of these factors with Pol II and chromatin. To elucidate the fate of the Paf1C when dissociated from Pol II, we examined the localization of the Paf1C components in paf1 and rtf1 mutant yeast strains. We found that although the Paf1C factors remain nuclear in paf1 and rtf1 strains, loss of Paf1 or Rtf1 results in a change in the subnuclear distribution of the remaining factors. In wild-type cells, Paf1C components are present in the nucleoplasm but not the nucleolus. In contrast, in both paf1 and rtf1 strains, the remaining factors are found in the nucleolus as well as the nucleoplasm. Loss of Paf1 affects nucleolar function; we observed that expression of MAK21 and RRP12, important for rRNA processing, is reduced concomitant with an increase in rRNA precursors in a paf1 strain. However, these changes are not the result of relocalization of the Paf1C because loss of Rtf1 does not cause similar changes in rRNA processing. Instead, we speculate that the change in localization may reflect a link between the Paf1C and newly synthesized mRNAs as they exit the nucleus.     

Alpha 1 adrenergic receptor control of renal blood vessels during aging

Aging humans and rats have a reduced renal vascular constriction response to stress, change in posture, or exercise. In this study, renal interlobar arteries from 9- (intermediate age) to 15-month-old (aging) male Wistar rats constricted less to alpha-adrenergic agonists than those of 4-month-old (young adult) rats. The reduced contraction to A61603 (alpha 1 A agonist) was similar to that to norepinephrine and phenylephrine. Therefore, it appears that the reduction in constriction is primarily related to alpha 1 A receptor stimulation. GeneChip microarray hybridization analysis of the interlobar arteries with the RAE 230A GeneChip indicated that there were no significant differences in gene expression for alpha 1 A/C, 1B, or 1D receptors between 4-month-old (young adult) and 1-year-old (aging) male Wistar rats. Competitive binding experiments (prazosin) revealed that maximal binding (Bmax, fmol/mg protein) of the alpha 1 receptors of interlobar arteries was reduced 25% by 10 months of age and 50% by 18+ months of age. Alpha 1 receptor-induced arterial constriction and prazosin binding were both down-regulated. The loss of receptor-initiated constriction likely includes down-regulation of maximum agonist binding by alpha 1 adrenergic receptors.     

Fetal rabbit pulmonary artery smooth muscle cell response to ryanodine is developmentally regulated

To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4-6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 microM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (K(Ca)) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and K(Ca) channels act together to control intracellular calcium only in fetal distal PASMCs.     

Early emergence in a butterfly causally linked to anthropogenic warming

There is strong correlative evidence that human-induced climate warming is contributing to changes in the timing of natural events. Firm attribution, however, requires cause-and-effect links between observed climate change and altered phenology, together with statistical confidence that observed regional climate change is anthropogenic. We provide evidence for phenological shifts in the butterfly Heteronympha merope in response to regional warming in the southeast Australian city of Melbourne. The mean emergence date for H. merope has shifted −1.5 days per decade over a 65-year period with a concurrent increase in local air temperatures of approximately 0.16°C per decade. We used a physiologically based model of climatic influences on development, together with statistical analyses of climate data and global climate model projections, to attribute the response of H. merope to anthropogenic warming. Such mechanistic analyses of phenological responses to climate improve our ability to forecast future climate change impacts on biodiversity.     

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic "Epsilonproteobacteria"

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the "Epsilonproteobacteria" (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.