Crystalline bacterial cell surface layers

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram-positive and Gram-negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S-layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S-layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram-negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad-application potential in biotechnology, vaccine development and molecular nanotechnology.     

Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue

Summary Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.Abbreviations BAP benzylaminopurine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - NOA naphthoxyacetic acid - PCR polymerase chain reaction - PVP polyvinylpyrrolidone - TDZ thidiazuron     

Restriction map of Chinese hamster mitochondrial DNA containing replication coordinates: comparison with Syrian hamster mitochondrial genome

A precise physical map, containing the structurally and operationally defined D-loop origin, terminal region, and direction of heavy-strand replication, has been constructed for mitochondrial DNA (mtDNA) from ovary (CHO-KI) and lung cells of Chinese hamster (Cricetulus griseus 2 N = 22), and compared with our previously established genome coordinates for mtDNA from Syrian hamster ( Mesocricetus auratus 2 N = 44). All four HpaI sites in Cricetulus are conserved in Mesocricetus (8 sites). Extensive variation exists for hexanucleotides cleaved by EcoRI HindIII PstI. KpnI and BamHI. Sequence divergence between Chinese and Syrian hamster mtDNAs, as reflected from analysis of the mapped recognition sites for these six endonucleases, is estimated as 5–9% base substitutions. mtDNAs from both hamster and several other mammalian species contain a commonly conserved HpaI site in the region of light strand initiation.     

Dicephalic — ADrosophila mutant affecting polarity in follicle organization and embryonic patterning

Summary The mutationdicephalic (dic) affects follicle development and thereby alters the antero-posterior polarity of embryonic patterning. It maps at a single locus (3–46.0±1.0) and can be characterized as a semi-dominant maternal effect mutation with low penetrance. Indic follicles, the 15 nurse cells form two clusters located at opposite poles of the oocyte; the numerical distribution of the nurse cells among the clusters varies from 7:8 to 1:14. Thedic egg shell carries a micropyle (anterior marker) at either pole, but the misshapen respiratory appendages are restricted to one of the two poles in most eggs. The malformed eggs rarely yield larvae and these are always abnormal anteriorly and/or posteriorly. The segment pattern expressed in their cuticle may represent two anterior parts of opposite polarities (double head type), two posterior parts of opposite polarities (double abdomen type, rare) or show uniform polarity. Lability of organization at the cystocyte stage appears as the primary developmental defect of the mutant.     

Inter-telomeric recombination is present in telomerase-positive human cells

Immortal cells require a mechanism of telomere length control in order to divide infinitely. One mechanism is telomerase, an enzyme that compensates the loss of telomeric DNA. The second mechanism is the alternative lengthening of telomeres (ALT) pathway. In ALT pathway cells, homologous recombination between telomeric DNA is the mechanism by which telomere homeostasis is achieved. We developed a novel homologous recombination reporter system that is able to measure inter-telomeric recombination in a sensitive manner. We asked the fundamental question if homologous recombination between different telomeres is present in telomerase-positive cells. In this in vitro study, we showed that homologous recombination between telomeres is detectable in ALT cells with the same frequency as in cells that utilize the telomerase pathway. We further described an ALT cell clone that showed peaks of recombination which were not detected in telomerase-positive clones. In telomerase-positive cells the frequency of inter-telomeric recombination was not increased by shortened telomeres or by a fragile telomere phenotype induced with aphidicolin. ALT cells, in contrast, responded to aphidicolin with an increase in the frequency of recombination. Our results indicate that inter-telomeric recombination is present in both pathways of telomere length control, but the factors that increase recombination are different in ALT and telomerase-positive cells.     

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.     

Complex Actions of Ionomycin in Cultured Cerebellar Astrocytes Affecting Both Calcium-Induced Calcium Release and Store-Operated Calcium Entry

The polyether antibiotic ionomycin is a common research tool employed to raise cytosolic Ca²⁺ in almost any cell type. Although initially thought to directly cause physicochemical translocation of extracellular Ca²⁺ into the cytosol, a number of studies have demonstrated that the mechanism of action is likely to be more complex, involving modulation of intrinsic Ca²⁺ signaling pathways. In the present study we assessed the effect of ionomycin on primary cultures of murine cerebellar astrocytes. Ionomycin concentrations ranging from 0.1 to 10 μM triggered a biphasic increase in cytosolic Ca²⁺, consisting of an initial peak and a subsequent sustained plateau. The response was dependent on concentration and exposure time. While the plateau phase was abolished in the absence of extracellular Ca²⁺, the peak phase persisted. The peak amplitude could be lowered significantly by application of dantrolene, demonstrating involvement of Ca²⁺-induced Ca²⁺-release (CICR). The plateau phase was markedly reduced when store-operated Ca²⁺-entry (SOCE) was blocked with 2-aminoethoxydiphenyl borate. Our results show that ionomycin directly affects internal Ca²⁺ stores in astrocytes, causing release of Ca²⁺ into the cytosol, which in turn triggers further depletion of the stores through CICR and subsequently activates SOCE. This mechanistic action of ionomycin is important to keep in mind when employing it as a pharmacological tool.     

Discovery of an intravenous hepatoselective glucokinase activator for the treatment of inpatient hyperglycemia

Glucokinase (hexokinase IV) continues to be a compelling target for the treatment of type 2 diabetes given the wealth of supporting human genetics data and numerous reports of robust clinical glucose lowering in patients treated with small molecule allosteric activators. Recent work has demonstrated the ability of hepatoselective activators to deliver glucose lowering efficacy with minimal risk of hypoglycemia. While orally administered agents require a considerable degree of passive permeability to promote suitable exposures, there is no such restriction on intravenously delivered drugs. Therefore, minimization of membrane diffusion in the context of an intravenously agent should ensure optimal hepatic targeting and therapeutic index. This work details the identification a hepatoselective GKA exhibiting the aforementioned properties.     

A Simple and Computationally Efficient Approach to Multifactor Dimensionality Reduction Analysis of Gene-Gene Interactions for Quantitative Traits

We present an extension of the two-class multifactor dimensionality reduction (MDR) algorithm that enables detection and characterization of epistatic SNP-SNP interactions in the context of a quantitative trait. The proposed Quantitative MDR (QMDR) method handles continuous data by modifying MDR’s constructive induction algorithm to use a T-test. QMDR replaces the balanced accuracy metric with a T-test statistic as the score to determine the best interaction model. We used a simulation to identify the empirical distribution of QMDR’s testing score. We then applied QMDR to genetic data from the ongoing prospective Prevention of Renal and Vascular End-Stage Disease (PREVEND) study.