Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts

Aprotinin, a bovine protease inhibitor of important therapeutic value, was expressed in tobacco plastid transformants. This disulphide bond-containing protein was targeted to the lumen of thylakoids using signal peptides derived from nuclear genes which encode lumenal proteins. Translocation was attempted via either the general secretion (Sec) or the twin-arginine translocation (Tat) pathway. In both cases, this strategy allowed the production of genuine aprotinin with its N-terminal arginine residue. The recombinant protease inhibitor was efficiently secreted within the lumen of thylakoids, accumulated in older leaves and was bound to trypsin, suggesting that the three disulphide bonds of aprotinin are correctly folded and paired in this chloroplast compartment. Mass spectrometric analysis indicated that translocation via the Sec pathway, unlike the Tat pathway, led predominantly to an oxidized protein. Translocation via the Tat pathway was linked to a slightly decreased growth rate, a pale-green leaf phenotype and supplementary expression products associated with the thylakoids.     

Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.     

热带季节性湿润林苦楝(Melia azedarach)径向生长季节动态及其对环境因子的响应

气候变化导致的温度升高和降水格局改变可能会影响到树木的生长速率和季节物候。西双版纳热带季节性湿润林分布在石灰岩山中部,属于热带喀斯特生境。由于土层浅薄,土壤保水能力极差,植物生长更容易遭到受到季节性干旱气候的影响。为探究热带季节性湿润林的树木径向生长季节动态及其对环境因子的响应,利用高精度树木生长仪连续两年监测了云南西双版纳热带季节性湿润林中落叶树种苦楝（Melia azedarach）的树干径向变化,并与同步监测的环境因子进行相关分析。结果表明,苦楝径向生长开始、结束以及持续生长的时间在年际间存在差异。与2018年相比,2019年苦楝生长开始和结束的时间较晚,且年生长量较小,这可能是与2019年雨季开始较晚且在生长季早期经历了严重的高温干旱有关。苦楝的径向日生长量与日降水量和相对湿度呈正相关关系,与光合有效辐射、水汽压亏缺和风速呈负相关关系,表明了在苦楝的径向生长主要受水分条件限制。在干旱年份（2019年）,苦楝的日生长量与降水和相对湿度的相关性更强。研究结果有助于进一步了解热带喀斯特生境树木生长对气候变化的敏感性以及树木适应季节性干旱气候的策略。     

Evaluation of microbial testing methods for the mutagenicity of quinoline and its derivatives

Using the mutational enhancement method and the Ames test, the mutagenicity and potential carcinogenicity of quinoline and its derivatives were determined and compared. Quinoline, 8-hydroxyquinoline, 5-hydroxyquinoline, 8-hydroxyquinoline sulfate, 6-nitroquinoline, 8-nitroquinoline, and 3-methylquinoline were mutagenic in the Ames direct plating test on TA 98 and TA 100 with activating system (S-9) from the rat liver. These compounds were not mutagenic in the mutational enchancement test onEscherichia coli HCR+ strain. 5,7-Dichloro-8-hydroxyquinoline, isoquinoline, and 2-chloroquinoline were nonmutagenic without or with S-9 in both the Ames and mutational enhancement test system. The compounds chloroquine, primaquine diphosphate, quinine hydrobromide, quinine hydrochloride, quinine lactate, quinine urea hydrochloride, quinine ethylcarbonate, quinine dihydrochloride, beta quinine quinine valerate, and quinine glycerophosphate were nonmutagenic with and without S-9 in the Ames test but mutagenic (20–60 g/ml) in the mutational enhancement test method onEscherichia coli HCR+ strains. The observations reported here point out that the Ames test responds negatively to several quinoline derivatives that are positive in the mutational enchancement test method.     

Complete Genome Sequence of the Facultative Anaerobic Magnetotactic Bacterium Magnetospirillum sp. strain AMB-1

Magnetospirillum sp. strain AMB-1 is a Gram-negative -proteobacteriumthat synthesizes nano-sized magnetites, referred to as magnetosomes,aligned intracellularly in a chain. The potential of this nano-sizedmaterial is growing and will be applicable to broad researchareas. It has been expected that genome analysis would elucidatethe mechanism of magnetosome formation by magnetic bacteria.Here we describe the genome of Magnetospirillum sp. AMB-1 wildtype, which consists of a single circular chromosome of 4967148bp. For identification of genes required for magnetosome formation,transposon mutagenesis and determination of magnetosome membraneproteins were performed. Analysis of a non-magnetic transposonmutant library focused on three unknown genes from 2752 unknowngenes and three genes from 205 signal transduction genes. Partialproteome analysis of the magnetosome membrane revealed thatthe membrane contains numerous oxidation/reduction proteinsand a signal response regulator that may function in magnetotaxis.Thus, oxidation/reduction proteins and elaborate multidomainsignaling proteins were analyzed. This comprehensive genomeanalysis will enable resolution of the mechanisms of magnetosomeformation and provide a template to determine how magnetic bacteriamaintain a species-specific, nano-sized, magnetic single domainand paramagnetic morphology.     

Labdane-type diterpenes: thermal effects on phospholipid bilayers, incorporation into liposomes and biological activity

Labd-13(E)-ene-8alpha,15-diol (1) and its derivative labd-13(E)-ene-8alpha-ol-15-yl-acetate (2) are water insoluble biological active molecules and their structures were elucidated using NMR and X-ray techniques. Differential scanning calorimetry (DSC) was applied to study the thermal effects of 1 and 2 on DPPC bilayers. Liposomes composed of egg phosphatidylcholine/dipalmytoylphosphatidylglycerol (9:0.1 molar ratio) were prepared by the thin-film hydration method and were used for incorporating 1 and 2. Free and liposomal 1 and 2 were tested for their activity against human cancer cell lines using the sulphorhodamine B assay. The effect of 1 and 2 on DPPC bilayers caused abolition of the pre-transition temperature, lowering of the main phase transition and reduction of the transition enthalpy only in the presence of cholesterol. The liposomes that have been designed and developed offer high incorporation efficiency; 62.4% (0.369 drug/lipid molar ratio) and 99.7% (0.661 drug/lipid molar ratio) for 1 and 2, respectively. Liposomal 2 showed growth-inhibiting activity against the majority of the tested cell lines.     

A new example of a tetranuclear iron(III) cluster containing the [Fe4O2] core: preparation, X-ray crystal structure, magnetochemistry and Mössbauer study of [Fe4O2(O2CMe)6(N3)2(phen)2]

Two synthetic procedures have been employed that allow access to the new tetranuclear cluster [Fe₄O₂(O₂CMe)₆(N₃)₂(phen)₂] (1), where phen is 1,10-phenanthroline. Complex 1 · 3MeCN displays an unusual structural asymmetry (observed for the second time) in its [Fe₄O₂]⁸⁺ core that can be considered as a hybrid of the bent (butterfly) and planar dispositions of four metal ions seen previously in such compounds with transition metals. Complex 1 has been characterized by variable-temperature magnetic susceptibility studies, and by IR and variable-temperature ⁵⁷Fe Mössbauer spectroscopies. Magnetochemical data reveal a diamagnetic ground state (S=0) with antiferromagnetic body-body and body-wingtip interactions between the iron(III) ions of the butterfly core (J_bb=−11 cm⁻¹, J_wb=−70 cm⁻¹). Magnetochemical and Mössbauer studies on 1 show that its structural asymmetry has practically no influence on these properties compared with the more symmetric types.     

Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy

Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.     

The evolutionary dynamics of endogenous retroviruses

Endogenous retroviruses (ERVs) are vertically transmitted intragenomic elements derived from integrated retroviruses. ERVs can proliferate within the genome of their host until they either acquire inactivating mutations or are lost by recombinational deletion. We present a model that unifies current knowledge of ERV biology into a single evolutionary framework. The model predicts the possible long-term outcomes of retroviral germline infection and can account for the variable patterns of observed ERV genetic diversity. We hope the model will provide a useful framework for understanding ERV evolution, enabling the testing of evolutionary hypotheses and the estimation of parameters governing ERV proliferation.     

2-micron circle plasmids do not reduce yeast life span

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir⁺ and cir⁰ strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.