Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.     

The p lambda CM system: phage immunity-specific incompatibility with IncP-1 plasmids

Summary A pCM2 replicon derived by an N^– deletion from ::Tn9 which carries the imm434 immunity region is incompatible with some (but not all) IncP-1 plasmids. The imm pCM1 replicon does not show the same incompatibility behavior.     

Evidence for interactions between the energy-dependent transport of sugars and the membrane potential in the yeastRhodotorula gracilis (Rhodosporidium toruloides)

Summary A membrane potential (inside negative) across the plasma membrane of the obligatory aerobic yeastRhodotorula gracilis is indicated by the intracellular accumulation of the lipid-soluble cations tetraphenylphosphonium and triphenylmethylphosphonium. The uptake of these ions is inhibited by anaerobic conditions, by uncouplers, by addition of diffusible ions, or by increase of the leakiness of the membrane caused by the polyene antibiotic nystatin. The membrane potential is strongly pH-dependent, its value increasing with decreasing extracellular proton concentration. Addition of transportable monosaccharides causes a depolarization of the electrical potential difference, indicating that the H⁺-sugar cotransport is electrogenic. The effect on the membrane potential is enhanced by increasing the sugar concentration. The half-saturation constants of depolarization ford-xylose andd-galactose were comparable to those of the corresponding transport system for the two sugars. All agents that depressed the membrane potential inhibited monosaccharide transport; hence the membrane potential provides energy for active sugar transport in this strain of yeast.     

Recurrent and founder mutations in the Netherlands—Phospholamban p.Arg14del mutation causes arrhythmogenic cardiomyopathy

Background

Recently, we showed that the c.40_42delAGA (p.Arg14del) mutation in the phospholamban (PLN) gene can be identified in 10–15 % of Dutch patients with dilated cardiomyopathy or arrhythmogenic cardiomyopathy. The arrhythmogenic burden of the p.Arg14del mutation was illustrated by the high rate of appropriate ICD discharges and a positive family history for sudden cardiac death.

Methods

Our goal was to evaluate the geographical distribution and the origin of this specific mutation in the Netherlands and to get an estimation of the prevalence in a Dutch population cohort. Therefore, we investigated the postal codes of the places of residence of PLN p.Arg14del mutation carriers and places of birth of their ancestors. In addition, a large population-based cohort (PREVEND) was screened for the presence of this mutation.

Results

By April 2012, we had identified 101 probands carrying the PLN p.Arg14del mutation. A total of 358 family members were also found to carry this mutation, resulting in a total of 459 mutation carriers. The majority of mutation carriers live in the northern part of the Netherlands and analysing their grandparents’ places of birth indicated that the mutation likely originated in the eastern part of the province of Friesland. In the PREVEND cohort we identified six heterozygous PLN p.Arg14del mutation carriers out of 8,267 subjects (0.07 %).

Conclusion

The p.Arg14del mutation in the PLN gene is the most frequently identified mutation in Dutch cardiomyopathy patients. The mutation that arose 575–825 years ago is likely to have originated from the eastern part of the province of Friesland and is highly prevalent in the general population in the northern part of the Netherlands.     

Indirect Estimates of Total Fertility Rate Using Child Woman/Ratio: A Comparison with the Bogue-Palmore Method

Indirect estimation methodologies of the total fertility rate (TFR) have a long history within demography and have provided important techniques applied demographers can use when data is sparse or lacking. However new methodologies for approximating the total fertility rate have not been proposed in nearly 30 years. This study presents a novel method for indirectly approximating the total fertility rate using an algebraic rearrangement of the general fertility rate (GFR) through the known relationship between GFR and TFR. It then compares the proposed method to the well-known Bogue-Palmore method. These methods are compared in 196 countries and include overall errors as well as characteristics of the countries that contribute to fertility behavior. Additionally, these methods were compared geographically to find any geographical patterns. We find this novel method is not only simpler than the Bogue-Palmore method, requiring fewer data inputs, but also has reduced algebraic and absolute errors when compared with the Bogue-Palmore method and specifically outperforms the Bogue-Palmore method in developing countries. We find that our novel method may be useful estimation procedure for demographers.     

Characterization and structure-guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436

One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non-activated C-H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β-ionone in non-allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure-guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non-allylic positions of β-ionone, allowing the simultaneous production of 2- and 3-hydroxy-β-ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole-cell system finally enabled the production of up to 0.5 g l⁻¹ hydroxylated products of β-ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.