Transcript profiling of terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell cultures

The understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseus, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseus cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol-4-phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macroarray, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseus comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseus cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseus cell cultures.     

Temperature-dependent STIM1 activation induces Ca²+ influx and modulates gene expression

Intracellular Ca(2+) is essential for diverse cellular functions. Ca(2+) entry into many cell types including immune cells is triggered by depleting endoplasmic reticulum (ER) Ca(2+), a process termed store-operated Ca(2+) entry (SOCE). STIM1 is an ER Ca(2+) sensor. Upon Ca(2+) store depletion, STIM1 clusters at ER-plasma membrane junctions where it interacts with and gates Ca(2+)-permeable Orai1 ion channels. Here we show that STIM1 is also activated by temperature. Heating cells caused clustering of STIM1 at temperatures above 35 °C without depleting Ca(2+) stores and led to Orai1-mediated Ca(2+) influx as a heat off-response (response after cooling). Notably, the functional coupling of STIM1 and Orai1 is prevented at high temperatures, potentially explaining the heat off-response. Additionally, physiologically relevant temperature shifts modulate STIM1-dependent gene expression in Jurkat T cells. Therefore, temperature is an important regulator of STIM1 function.     

Increased hydrophobicity and decreased backbone flexibility explain the lower solubility of a cataract-linked mutant of γD-crystallin

A number of point mutations in γD-crystallin are associated with human cataract. The Pro23-to-Thr (P23T) mutation is perhaps the most common, is geographically widespread, and presents itself in a variety of phenotypes. It is therefore important to understand the molecular basis of lens opacity due to this mutation. In our earlier studies, we noted that P23T shows retrograde and sharply lowered solubility, most likely due to the emergence of hydrophobic patches involved in protein aggregation. Binding of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate (Bis-ANS) dye (a probe commonly used for detecting surface hydrophobicity) competed with aggregation, suggesting that the residues involved in Bis-ANS binding are also involved in protein aggregation. Here, using NMR spectroscopy in conjunction with Bis-ANS binding, we identify three residues (Y16, D21, and Y50) in P23T that are involved in binding the dye. Furthermore, using ¹⁵N NMR relaxation experiments, we show that, in the mutant protein, backbone fluctuations are restricted to the picosecond-to-nanosecond and microsecond timescales relative to the wild type. Our present studies specify the residues involved in these two pivotal characteristics of the mutant protein, namely increased surface hydrophobicity and restricted mobility of the protein backbone, which can explain the nucleation and further propagation of protein aggregates. Thus, we have now identified the residues in the P23T mutant that give rise to novel hydrophobic surfaces, as well as those regions of the protein backbone where fluctuations in different timescales are restricted, providing a comprehensive understanding of how lens opacity could result from this mutation.     

The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protoplasts

Brassica napus L.(cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid protoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected lines produced selfed progeny which were tolerant to from 10–100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progeny of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the AHAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.     

Structural assembly of two-domain proteins by rigid-body docking

Background  

Modelling proteins with multiple domains is one of the central challenges in Structural Biology. Although homology modelling has successfully been applied for prediction of protein structures, very often domain-domain interactions cannot be inferred from the structures of homologues and their prediction requiresab initiomethods. Here we present a new structural prediction approach for modelling two-domain proteins based on rigid-body domain-domain docking.     

Primary ciliary dyskinesia (Siewert's / Kartagener's Syndrome): Respiratory symptoms and psycho-social impact

Background

Although the pathophysiological defect in primary ciliary dyskinesia (PCD; Siewert's / Kartagener's syndrome) is now well characterised, there are few studies of the impact of the condition upon health function, particularly in later life. This study assesses the health impact of the condition in a large group of patients. In addition, it assesses the similarity in age of diagnosis, symptoms and problems of those with situs inversus (PCD-SI) and those with situs solitus (PCD-SS).

Methods

Postal questionnaire sent to members of the UK Primary Ciliary Dyskinesia Family Support Group. The questionnaire contained the St. George's Respiratory Questionnaire (SGRQ) and the SF-36 questionnaire for assessing health status.

Results

93 questionnaires were returned, representing a 66% response rate. Replies were received from similar numbers of PCD-SI and PCD-SS. Individuals with PCD-SI did not show a significant tendency to be diagnosed earlier, and neither did they show any difference in their symptoms, or the relationship of symptoms to age. Respiratory symptoms were fairly constant up until the age of about 25, after which there was a slow increase in symptoms, and a decline in health status, patients over the age of 40 being about one and a half standard deviations below the mean on the physical component score of the PCS. Patients diagnosed earlier in life, and hence who had received more treatment for their condition, had better scores on the SGRQ Impact and Activity scores.

Conclusions

PCD is a chronic condition which has a progressively greater impact on health in the second half of life, producing significant morbidity and restriction of life style. Early diagnosis, and hence earlier treatment, may improve symptoms and the impact of the condition.     

Growth and terpenoid indole alkaloid production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri T_L-DNA and T_R-DNA genes, there were only three different categories of independent hairy root clones—C1 (rolA&B ⁺/ags ⁺), C2 (rolA&B ^-/ags ⁺) and C3 (rolA&B ⁺/ags ^–). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T_L-DNA and T_R-DNA genes (C1) and the T_L-DNA gene (C3) alone. Two rolA&B ^– but ags ⁺ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the T_L-DNA and T_R-DNA genes on growth and alkaloid accumulation in these root clones is discussed.Abbreviations ags Agropine synthase - Ri Root-inducing - T _L -DNA Left-terminus DNA - T _R -DNA Right-terminus DNA - TIAs Terpenoid indole alkaloids     

Effects of Abiotic Factors on the Phylogenetic Diversity of Bacterial Communities in Acidic Thermal Springs

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E_h of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.