Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

The origin of polyploid genomes of bluegrasses Poa L. and gene flow between Northern Pacific and sub-Antarctic islands

The involvement of present-day diploid bluegrass species in the formation of polyploid genomes was investigated using comparison of sequences of internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA sequence. It was demonstrated that highly polyploid New Zealand bluegrasses, P. cita (2n = 84; ca. 96 to 100), P. chathamica (2n = 112), and P. litorosa (2n = 263 to 266) formed separate highly supported clade together with tetraploids (2n = 28) P. intrusa, P. anceps, and P. trioides (Austrofestuca littoralis). Among the diploid species (2n = 14), the closest relatives of these species, as well as of the polyploid species of section Poa, are the genomes of Eurasian species P. remota, P. chaixcii (sect. Homalopoa), P densa (Bolbophorum), and P. sibirica (sect. Macropoa). Nuclear genomes of polyploid Stenopoa, Tichopoa, Oreinos, and Secundae are definitely related to the genome of Arctic species P. pseudabbreviata (sect. Abbreviatae). On the contrary, judging by the genes for nuclear 45S rRNA, genomes of diploid P. trivialis (sect. Pandemos), P. annua, and P. supina (sect. Ochlopoa both) are only remotely related to the genomes of highly polyploid species (distances p between them and other bluegrass species from different sections of subgenus Poa constitute 6-10% and 11-15%, respectively). The conclusion on the relationships between highly polyploid and diploid bluegrass species was tested using analysis of synapomorphic mutations in the 5.8S rRNA gene. It was demonstrated that genomes of Poa eminens (2n = 42) and P. schischkinii (2n = 70) (sect. Arctopoa both) were noticeably different in ITS regions from the genomes of the members of the type subgenus Poa. A comparison of the Arctopoa ITS regions showed that the differences between them constituted only 0.2%. At the same time, p distances between the Arctopoa ITS and those from the species belonging to other sections of the genus Poa varied from 5 to 14%. South American species P chonotica (sect. Andinae) (=Ncoraepoa chonotica) (2n = 42) was found to be related to Arctagrostis, Festucella, and Hookerochloa, being at the same time quite distant from the other species of the genus Poa. Polymorphic in chromosome number highly polyploid species of Northern Hemisphere, P. arctica (2n = 42 to 106), P. turneri (2n = 42, 63 to 64), and P. smirnovii (2n = 42, 70) (sect. Malacanthae) are relative to a large group of tetraploid (2n = 28) endemic bluegrass species from New Zealand and sub-Antarctic islands (P. novae-zelandiae and allied species).     

Tagaturonate–fructuronate epimerase UxaE,a novel enzyme in the hexuronate catabolic network in Thermotoga maritima

Thermotoga maritima is a marine hyperthermophilic microorganism that degrades a wide range of simple and complex carbohydrates including pectin and produces fermentative hydrogen at high yield. Galacturonate and glucuronate, two abundant hexuronic acids in pectin and xylan, respectively, are catabolized via committed metabolic pathways to supply carbon and energy for a variety of microorganisms. By a combination of bioinformatics and experimental techniques we identified a novel enzyme family (named UxaE) catalysing a previously unknown reaction in the hexuronic acid catabolic pathway, epimerization of tagaturonate to fructuronate. The enzymatic activity of the purified recombinant tagaturonate epimerase from T. maritima was directly confirmed and kinetically characterized. Its function was also confirmed by genetic complementation of the growth of the Escherichia coli uxaB knockout mutant strain on galacturonate. An inferred novel galacturonate to mannonate catabolic pathway in T. maritima was reconstituted in vitro using a mixture of recombinant purified enzymes UxaE, UxaC and UxuB. Members of the newly identified UxaE family were identified in ～ 50 phylogenetically diverse heterotrophic bacteria from aquatic and soil environments. The genomic context of respective genes and reconstruction of associated pathways suggest that UxaE enzymatic and biological function remains conserved in all of these species.     

Is low dose inhaled corticosteroid therapy as effective for inflammation and remodeling in asthma? A randomized,parallel group study

Background

While most of the clinical benefits of inhaled corticosteroid (ICS) therapy may occur at low doses, results of dose-ranging studies are inconsistent. Although symptom/lung function response to low and high dose ICS medication is comparable, it is uncertain whether low dose ICSs are as effective as high dose in the treatment of inflammation and remodeling.

Methods

22 mild or moderate asthmatic adult subjects (corticosteroid free for > 2 months) participated in a randomized, parallel group study to compare effects of fluticasone propionate (FP) 200 mcg/day and 1000 mcg/day. Alveolar macrophage (AM)-derived cytokines and basement membrane thickness (BMT) were measured at baseline and after 7 weeks treatment while symptoms, spirometry, exhaled nitric oxide (eNO) and airway hyperresponsiveness (AHR) to mannitol at baseline and 6 weeks.

Results

FP improved spirometry, eNO, symptoms and AHR with no difference between low and high dose FP. Both high and low dose FP reduced GM-CSF, TNF-alpha and IL-1ra, with no change in BMT and with no differences between low and high dose FP.

Conclusions

200 μg/day of FP was as effective as 1000 μg/day in improving asthma control, airway inflammation, lung function and AHR in adults in the short term. Future studies should examine potential differential effects between low and high dose combination therapy (ICS/long acting beta agonist) on inflammation and airway remodeling over longer treatment periods.     

Chromosome Maps of Trilliaceae: II. A Study of the Genome Composition in Polyploid Species of the Genus Trillium by Fluorescence Nucleotide Base-Specific Staining of Heterochromatic Chromosome Regions

Chromosome banding with nucleotide base-specific fluorochromes chromomycin A₃ (CMA) and Hoechst 33258 (H33258) was used to study the karyotypes and to construct cytological maps for diploidTrillium camschatcense(2n = 10), tetraploid T. tschonoskii(2n = 20), hexaploidT. rhombifolium (2n = 30), and a triploid T. camschatcense × T. tschonoskii hybrid (T. × hagae, 2n = 15). With H33258, species- and genome-specific patterns with numerous AT-rich heterochromatin bands were obtained for each of the four forms; CMA revealed a few small, mostly telomeric GC-rich bands. In T. tschonoskii, the two subgenomes were similar to each other and differed from the T. camschatcense genome; on this evidence, the species was considered to be a segmental allotetraploid. InT. ×hagae, one T. camschatcense and both T. tschonoskii subgenomes were identified. The subgenomes of T. rhombifoliumonly partly corresponded to the T. camschatcense and T. tschonoskii genomes, in contrast to the morphologically identical Japanese species T. hagae. This was assumed to indicate that allohexaploids T. rhombifolium and T. hagae originated independently at different times; i.e., their origin is polyphyletic. Based on the chromosome maps, a new nomenclature was proposed for theTrillium genomes examined: K₁K₁ for T. camschatcense,T₁T₁T₂T₂ for T. tschonoskii,K₁T₁T₂ for T. × hagae, and K_1RK_1RT_1RT_1RT_2RT_2R for T. rhombifolium.     

Redistribution of subpopulations of peritoneal macrophages with Fc gamma receptors under the effect of an immunomodulator

The heterogeneity of resident peritoneal macrophages and peritoneal macrophages was studied in different periods following oral administration of sodium nucleinate according to their ability to bind and phagocytize sheep erythrocytes opsonized by means of specific rabbit IgG. Using a mathematical method developed earlier, it has been possible to demonstrate that resident peritoneal macrophages can be divided into two subpopulations--actively and poorly binding macrophages but, after activation by sodium nucleinate--into three subpopulations. Fractionation of the macrophages according to their ability to adhesion within a temperature gradient has shown that the same peaks are traced in the fractions as in the overall pool of cells, but in different quantitative ratios. It has also been demonstrated that phagocytic activity is reduced in macrophages capable of adhesion to plastic at lower temperatures.     

Influence of the annual flood-pulse on catch per unit effort,condition and reproduction of Clarias gariepinus from the upper Okavango Delta,Botswana

Catch per unit effort (CPUE), length, weight and maturity data for Clarias gariepinus were collected during monthly gillnet surveys in the upper Okavango Delta between 2001 and 2009 to investigate their relationship with the annual flood-pulse. CPUE, condition factor (K) and the proportion of ripe-running fish (P_RR) in the population followed a unimodal annual cycle that could be modelled using water temperature and flood-pulse hydrology. Increased CPUE during declining water levels was most likely a result of feeding migrations and aggregation behaviour. The observed increase in K during low floods in October and November preceded the increase in P_RR, which increased mainly with increasing temperature but appeared less dependent on flow. This study provided quantitative evidence that the biology of fish in the Okavango Delta is mainly dependent on the annual flood regime and, therefore, that conservation efforts should be focused on maintaining natural flow patterns in the face of climate change and potential water extraction schemes upstream.