Molecular cloning and expression analyses of a new gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Taxus × media

A new full-length cDNA encoding 3-hydroxy-3-methylglutaryl-CoA synthase (designated as TmHMGS, GenBank Accession No. AY644708), which catalyses the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the taxol biosynthetic pathway, was isolated from young leaves of Taxus × media by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of TmHMGS contained a 1431 bp open reading frame (ORF) encoding a deduced protein of 476 amino acid residues. The deduced protein had an isoelectric point of 5.23 and a calculated molecular mass of about 53 kDa. Amino acid sequence comparison analysis showed that TmHMGS had high similarity with a number of HMGSs ranging from Schizosaccharomyces pombe to humans, with much higher identity with other HMGSs from plants than those from yeast and humans. Phylogenic analysis showed that TmHMGS had closest relationship with HMGS from Pinus sylvestris. Tissue expression pattern analysis showed that TmHMGS expressed in needles and stems at similar level, but no expression could be detected in roots. Expression of TmHMGS was all induced by under different elicitors such as silver nitrate, ammonium ceric sulphate and methyl jasmonate, revealed that TmHMGS was an elicitor-responsive gene.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.     

G2 arrest, binucleation, and single-parameter DNA flow cytometric analysis

One important facet of flow cytometry involves the effects of pharmacological agents on cell cycle progression. Comparative G2 fraction perturbations were examined: effects of sodium butyrate on articular chondrocytes, effects of an antineoplastic agent (SOAZ) and an antirheumatic drug (D-penicillamine) on HeLa cells. Even though DNA flow cytometric analysis detects preferentially an induction of G2 arrest, the mode of action of these agents on the cell cycle is different. Sodium butyrate and D-penicillamine lead to an increase of binucleate cells due to cytokinesis perturbation. Because of similar fluorescence intensity, distinguishing G2 from binucleate GO/1 cells is not easily possible using DNA content measurement and reflects a failure of flow cytometry in the detection of binucleate cells. Rapid cell cycle analysis of single cells should contribute greatly to the study of pharmacological interactions, but DNA flow cytometric measurements obtained from cultured cells exposed to certain agents must be cautiously interpreted because those may interact on cytokinesis and induce artefacts in histogram interpretation.     

The kic1 kinase of schizosaccharomyces pombe is a CLK/STY orthologue that regulates cell-cell separation

The CLK/STY kinases are a family of dual-specificity protein kinases implicated in the regulation of cellular growth and differentiation. Some of the kinases in the family are shown to phosphorylate serine-arginine-rich splicing factors and to regulate pre-mRNA splicing. However, the actual cellular mechanism that regulates cell growth, differentiation, and development by CLK/STY remains unclear. Here we show that a functionally conserved CLK/STY kinase exists in Schizosaccharomyces pombe, and this orthologue, called Kic1, regulates the cell surface and septum formation as well as a late step in cytokinesis. The Kic1 protein is modified in vivo, likely by phosphorylation, suggesting that it can be involved in a control cascade. In addition, kic1(+) together with dsk1(+), which encodes a related SR-specific protein kinase, constitutes a critical in vivo function for cell growth. The results provide the first in vivo evidence for the functional conservation of the CLK/STY family through evolution from fission yeast to mammals. Furthermore, since cell division and cell-cell interaction are fundamental for the differentiation and development of an organism, the novel cellular role of kic1(+) revealed from this study offers a clue to the understanding of its counterparts in higher eukaryotes.     

Predictive systems ecology

Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.     

Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed diets with low or high phytate content

A six-day experiment was conducted to estimate the relative bioavailability values (RBV) of zinc (Zn) in three organic sources (oZn) with different chelation strengths compared to inorganic ZnSO₄ (iZn) for broilers fed a low or high phytate diet. A total of 1080, one-d-old male broiler chicks were randomly assigned to one of 18 dietary treatments (six replicates cages of ten chicks per cage) in a completely randomized design involving a 2 × 2 × 4 factorial arrangement with two levels of added phytate (0 or 10 g phytate as sodium phytate/kg), two levels of added Zn (30 or 60 mg/kg) and four Zn sources (iZn and three oZn sources) plus one low and one high phytate control treatments without Zn addition. The three oZn sources consisted of (1) Zn amino acid with weak chelation strength (ZnAA-L, formation quotient Q_f = 6.6, containing 119 g Zn/kg), (2) Zn proteinate with moderate chelation strength (ZnPRO-M, Q_f = 30.7, containing 133 g Zn/kg) or (3) Zn proteinate with strong chelation strength (ZnPRO-H, Q_f = 944.0, containing 186 g Zn/kg). Chicks were harvested at 6 days of age and pancreas metallothionein (MT) mRNA expression was used to estimate Zn RBV. Pancreas MT mRNA expression increased (P<0.01) as dietary Zn level increased. Chicks fed high phytate diets had lower (P<0.05) MT mRNA expression than chicks fed low phytate diets. Based on multiple linear regression slope ratios with ZnSO₄ set at 1.00, the RBV of ZnAA-L, ZnPRO-M and ZnPRO-H were 1.01, 1.28 and 0.70, respectively, for low phytate diets, and 1.05, 1.39 and 0.92, respectively, for high phytate diets. The slope for the oZn source with moderate chelation strength differed (P<0.05) from iZn and the other two oZn sources. The RBV of ZnAA-L, ZnPRO-M and ZnPRO-H under the high phytate diet increased by 0.04, 0.11 and 0.22, respectively, compared to those under the low phytate diet. Results indicate that the oZn sources with moderate or strong chelation strength offer partial or complete resistance to interference from high dietary phytate during digestion; and the oZn with moderate chelation strength had a greater RBV with both low and high phytate diets than iZn or oZn sources with weak or strong chelation strength.     

Atrial Arrhythmia in Ageing Spontaneously Hypertensive Rats: Unraveling the Substrate in Hypertension and Ageing

Background

Both ageing and hypertension are known risk factors for atrial fibrillation (AF) although the pathophysiological contribution or interaction of the individual factors remains poorly understood. Here we aim to delineate the arrhythmogenic atrial substrate in mature spontaneously hypertensive rats (SHR).

Methods

SHR were studied at 12 and 15 months of age (n = 8 per group) together with equal numbers of age-matched normotensive Wistar-Kyoto control rats (WKY). Electrophysiologic study was performed on superfused isolated right and left atrial preparations using a custom built high-density multiple-electrode array to determine effective refractory periods (ERP), atrial conduction and atrial arrhythmia inducibility. Tissue specimens were harvested for structural analysis.

Results

Compared to WKY controls, the SHR demonstrated: Higher systolic blood pressure (p<0.0001), bi-atrial enlargement (p<0.05), bi-ventricular hypertrophy (p<0.05), lower atrial ERP (p = 0.008), increased atrial conduction heterogeneity (p = 0.001) and increased atrial interstitial fibrosis (p = 0.006) & CD68-positive macrophages infiltration (p<0.0001). These changes resulted in higher atrial arrhythmia inducibility (p = 0.01) and longer induced AF episodes (p = 0.02) in 15-month old SHR. Ageing contributed to incremental bi-atrial hypertrophy (p<0.01) and atrial conduction heterogeneity (p<0.01) without affecting atrial ERP, fibrosis and arrhythmia inducibility. The limited effect of ageing on the atrial substrate may be secondary to the reduction in CD68-positive macrophages.

Conclusions

Significant atrial electrical and structural remodeling is evident in the ageing spontaneously hypertensive rat atria. Concomitant hypertension appears to play a greater pathophysiological role than ageing despite their compounding effect on the atrial substrate. Inflammation is pathophysiologically linked to the pro-fibrotic changes in the hypertensive atria.     

Identification of quantitative trait loci for shank length and growth at different development stages in chicken

Shank length affects chicken leg health and longer shanks are a source of leg problems in heavy-bodied chickens. Identification of quantitative trait loci (QTL) affecting shank length traits may be of value to genetic improvement of these traits in chickens. A genome scan was conducted on 238 F₂ chickens from a reciprocal cross between the Silky Fowl and the White Plymouth Rock breeds using 125 microsatellite markers to detect static and developmental QTL affecting weekly shank length and growth (from 1 to 12 weeks) in chickens. Static QTL affected shank length from birth to time t , while developmental QTL affected shank growth from time t− 1 to time t . Seven static QTL on six chromosomes (GGA2, GGA3, GGA4, GGA7, GGA9 and GGA23) were detected at ages of 2, 3, 4, 5, 6, 7, 9 and 12 weeks, and six developmental QTL on five chromosomes (GGA1, GGA2, GGA4, GGA5 and GGA23) were detected for five shank growth periods, weeks 2–3, 4–5, 5–6, 10–11 and 11–12. A static QTL and a developmental QTL ( SQSL1 and DQSL2 ) were identified at GGA2 (between ADL0190 and ADL0152 ). SQSL1 explained 2.87–5.30% of the phenotypic variation in shank length from 3 to 7 weeks. DQSL2 explained 2.70% of the phenotypic variance of shank growth between 2 and 3 weeks. Two static and two developmental QTL were involved chromosome 4 and chromosome 23. Two chromosomes (GGA7 and GGA9) had static QTL but no developmental QTL and another two chromosomes (GGA1 and GGA5) had developmental QTL but no static QTL. The results of this study show that shank length and shank growth at different developmental stages involve different QTL.     

Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter-retrotransposon amplification polymorphism markers

The genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.