Shengmai Formula Ameliorates Pathological Characteristics in AD <Emphasis Type="Italic">C.</Emphasis> <Emphasis Type="Italic">elegans</Emphasis>

Shengmai (SM) formula, a classical traditional Chinese medicine formula, is composed of Panax ginseng (Pg), Ophiopogon japonicus (Oj), and Schisandra Chinesis (Sc). SM has been clinically used to treat heart failure and ischemic heart disease. Although SM formula has been reported to be potential for fighting against Alzheimer’s disease (AD) by previous works, there are many gaps in our knowledge on its usage in AD treatment on an organism level and will then need to be further clarified. In this study, transgenic Caenorhabditis elegans expressing human Aβ_1–42 are used to evaluate SM formula efficacy to treat AD phenotype and to investigate its underlying mechanism. The results showed that SM formula ameliorated AD pathological characteristics of paralysis behavior and chemotaxis defect in transgenic C. elegans. With SM treatment, the number of Aβ deposits decreased, the levels of gene expressions of hsp16-2, hsp16-41, ace-1, ace-2, and TNFA1P1 homolog genes were down-regulated. Our results also showed that Oj exhibited more stronger effect on delaying paralysis in worms than Pg and Sc did, and synergistic action was observed between Pg and Oj, and Sc further enhanced the activity of Pg/Oj combination on delaying paralysis behavior. Further, SM with herbs of Pg, Oj, and Sc at a dose proportion of 9:9:6 exhibited superior therapeutic efficacy in comparison with herbs at other dose proportions. After SM formula extracted by ethanol, it delayed AD symptoms on a wider dose from 0.2 to 10.0 mg/mL with no toxic effect. These results provided more evidence for SM formula being potential to be used to treat AD.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Effect of Phenolic Compounds Against A<Emphasis Type="Italic">β</Emphasis> Aggregation and A<Emphasis Type="Italic">β</Emphasis>-Induced Toxicity in Transgenic <Emphasis Type="Italic">C. elegans</Emphasis>

Substantial evidence suggests that the aggregation of amyloid-β (Aβ) peptide into fibrillar structures that is rich in β-sheets is implicated as the cause of Alzheimer’s disease. Therefore, an attractive therapeutic strategy is to prevent or alter Aβ aggregation. Phenolic compounds are natural substances that are composed of one or more aromatic phenolic rings and present in wine, tea, fruits, vegetables and a wide variety of plants. In this work, we investigated the effects of ferulic acid, morin, quercetin and gossypol against Aβ aggregation. From the ThT and turbidity assays, it is observed that in addition to the fibril aggregate, another type of aggregate is formed in the presence of morin, quercetin, and gossypol. On the other hand, ferulic acid did not prevent fibril formation, but it did appear to reduce the average length of fibrils compared to Aβ alone. To study the protective effects of phenolic compounds on Aβ-induced toxicity, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism, human Aβ is expressed intracellularly in the body wall muscle. We found that exposure of Caenorhabditis elegans to ferulic acid give more protection against Aβ toxicity than morin, quercetin and gossypol.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

AFLP and SSR polymorphism in a <Emphasis Type="Italic">Coffea</Emphasis> interspecific backcross progeny [(<Emphasis Type="Italic">C. heterocalyx</Emphasis> × <Emphasis Type="Italic">C. canephora</Emphasis>) × <Emphasis Type="Italic">C. canephora</Emphasis>]

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome (Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes (Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.Communicated by H.F. Linskens     

<Emphasis Type="Italic">Luteoamylascus aculeatus</Emphasis> (Pezizomycetes,Pezizaceae): a new genus and species near the <Emphasis Type="Italic">Pachyphlodes</Emphasis>–<Emphasis Type="Italic">Amylascus</Emphasis> lineage

The new taxon Luteoamylascus aculeatus described in this article is proposed to accommodate two collections of a hypogeous ascomycete from central Spain, characterized by a tomentose yellowish peridium, labyrinth-like gleba filled with whitish hyphae, and intensely reacting amyloid asci. ITS, 28S, and RPB2 data suggest that this new taxon is an independent lineage proposed here as the new genus Luteoamylascus. Until now, this lineage was only known from ectomycorrhizal root tips and mitotic spore mats. In phylogenetic analyses, the Luteoamylascus lineage is placed close to the genera Amylascus, Pachyphlodes, and Scabropezia. Morphological data suggest an affinity with Amylascus.     

Micro-NMR elucidates altered metabolites in the Parkinson’s disease-related <Emphasis Type="Italic">catp-6</Emphasis> genotype of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Introduction

A severe form of Parkinson’s disease (PD) is the Kufor-Rakeb syndrome. Here mutations in the ATP13A2 (PARK9) gene lead to an early juvenile-onset Parkinsonism often accompanied by dementia. ATP13A2 encodes a lysosomal P-type ATPase. Its ortholog in Caenorhabditis elegans is the catp-6 gene where phenotypes with mutations in the alleles ok3473 and tm3190 show high mortality and low reproduction.

Objectives

Since PD is difficult to study in humans we wanted to investigate the potential to use C. elegans as model for the Kufor-Rakeb syndrome. As it is difficult to obtain enough catp-6 mutant worms for standard NMR metabolic profiling, we explored focused ultrasonication extraction and miniaturized NMR as techniques to overcome this limitation.

Methods

One- and two-dimensional NMR experiments (¹ H, JRES, TOCSY) were performed with a commercial high-resolution magic angle spinning (HR-MAS) probe (25 µL sample volume). Significant features were identified through analysis of variance (ANOVA, p?<?0.05), volcano plots (p?<?0.05, fold change?>1.5), PCA, and PLS-DA.

Results

Assignment of statistically relevant peaks resulted in the identification of twenty altered metabolites. Previous studies on catp-6 mutants identified strong morphological and functional changes in their mitochondria. Our findings of altered TCA metabolites (fumarate, succinate), branched-chain amino acids (leucine, isoleucine and valine) and nucleotides (AMP, ATP and GTP), formate and hypoxanthine appear to support these findings. Highest fold changes (< ?5) in wildtype relative to both catp-6 strains were found for GTP. Formic acid is known to inhibit the mitochondrial respiratory chain complex IV and high hypoxanthine in catp-6 indicates an increased nucleotide salvage pathway. Alterations in most of the remaining metabolites may be the result of the recently discovered activation of AMPK (AMP-activated protein kinase) and inhibition of mTOR (mechanistic target of rapamycin) pathways together with a catabolic response to recover energy production.

Conclusions

If the effect of the catp-6 mutation in C. elegans at the level of metabolites is correlated to the metabolic dysfunction in the human PARK9 ortholog, then it may be possible to uncover the molecular mechanism behind Parkinsonism and the Kufor-Rakeb syndrome.

     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

Comparative Functional Analysis of Rat <Emphasis Type="Italic">TGF-β1</Emphasis> and <Emphasis Type="Italic">Xenopus laevis</Emphasis><Emphasis Type="Italic">TGF-β5</Emphasis> Promoters Suggest Differential Regulations

We have carried out a comparative functional analysis of the rat TGF-1 and Xenopus laevis TGF-5 promoters across several mammalian and amphibian cell lines. Progressive deletion constructs of both the promoters have been made using a PCR based approach and the basal promoter activities studied in Xenopus tadpole cell line (XTC), Xenopus adult kidney fibroblast cell line (A6), human hepatoma cell line (HepG2), normal rat kidney cell line (NRK), and Chinese hamster ovary cell line (CHO). Data suggests that the basal promoter activity of TGF-1 is low as compared to TGF-5 promoter in XTC cells but comparable in A6 cells, while TGF-5 promoter shows nearly negligible activity as compared to TGF-5 promoter in all the tested mammalian cell lines. Moreover, TGF-5 promoter is found to be repressed in XTC cells on treatment with TGF-5 protein. Thus, the regulation of TGF-1 and TGF-5 promoters is distinct in amphibian and mammalian species. We therefore suggest that contrary to the suggested functional equivalence of TGF-1 and TGF-5 proteins, TGF-1 and TGF-5 genes have distinct functions in their respective species. Present address (Kartiki V. Desai): Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH Bldg 41, Room C619, Bethesda, MD 20892, USA     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.