Improved protoplast culture and agarose media

Media solidified with agarose resulted in higher plating efficiency of protoplasts than commonly used agar media. Improved culture efficiency was observed with mesophyll protoplasts of Nicotiana tabacum and N. plumbaginifolia and with protoplasts isolated from cell lines of Daucus carota, Hyoscyamus muticus and two lines of N. tabacum. The improvement with agarose was consistent over a wide cell density range and also for different media. The positive effect was not due to the lower temperature at which the protoplasts could be plated. Culture experiments with mixtures of different agar types generally gave intermediate division frequencies. There was no obvious effect on plating efficiency in experiments where diffusion was permitted between agar and other agar types.     

A rapid assay for genetic complementation of nitrate reductase deficiency via bulk protoplast fusion

Summary Genetic complementation of different nitrate reductase-deficient (NR^-) Nicotiana plumbaginifolia cell lines could be recognized in the described fusion disc technique as early as 5–10 days after protoplast fusion. Protoplasts of previously characterized mutant cell lines, belonging to four different complementation groups, were mixed in pairwise combinations and fused by the drop-wise fusion technique at very high protoplast densities (10⁶ protoplasts in an 8–10 mm spot) which resulted in the formation of a fusion disc on the bottom of the petri dish. After 5–10 days of incubation in K3 medium the restoration of NR activity could be detected directly in the original culture by the in vivo NR assay. Such a rapid test giving information about the genetic complementation of different auxotrophic cell lines has not previously been published for plants.     

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5-³H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5-³H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.     

Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation

Protoplasts of the nicotinamide-deficient Hyoscyamus muticus cell line nic^– IVH2 and of the nitrate reductase-deficient Nicotiana tabacum cell line NR^– cnx 68 were induced to fuse. Selection for putative interspecific hybrid clones was via auxotroph complementation. Controls included tests for cross-feeding, cross inhibition, PEG-induced variation, culture-induced variation, reversion, viability, delayed selection. Protrotrophic cell lines were recovered exclusively from PEG-treated mixtures of both protoplast types. The putative hybrid clones grew independent of exogenously supplied auxins and cytokinins, and at a faster rate than either parent. The morphogenic potential of different clones varied from non-morphogenic to regeneration of fertile plants. Indirect evidence for the hybrid nature of the clones is provided from a) tight selection, b) hormone-independent growth, c) hybrid vigour, d) extreme morphological variation, e) isoenzyme bands from both parents, f) morphogenic potential. Definite proof for the hybrid nature was, however, provided from species-specific DNA hybridization. Chimerism could be excluded since only the large subunit of Hyoscyamus muticus ribulose bisphosphate carboxylase was found and since species-specific DNA hybridization identified clones which gave no Nicotiana tabacum signal.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BAP 6-Benzylaminopurine - PEG Polyethyleneglykol - MES (2[N-morpholino]ethane sulfonic acid - Tris-HCl Tris(hydroxymethyl)-aminoethane-HCl % is given throughout in w/v - FMI Friedrich Miescher-Institut     

MicroRNA 520d-3p inhibits gastric cancer cell proliferation,migration, and invasion by downregulating EphA2 expression

Aberrant expression of microRNAs (miRNAs) has been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the roles of miR-520d-3p on gastric cancer (GC) cell proliferation, migration, and invasion, and confirmed that this miRNA regulates EphA2 expression. The mRNA expression levels of miR-520d-3p and EphA2 in GC tissues and cell lines were evaluated. The clinical and prognostic significance of miR-520d-3p was assessed. The biological function of miR-520d-3p in GC cells was investigated using a methylthiazolyldiphenyl-tetrazolium bromide assay, cell cycle assay, transwell invasion assay, and wound-healing assay. miR-520d-3p expression was down-regulated and inversely correlated with the expression of EphA2 in GC tissues and cell lines. Lower expression of miR-520d-3p was associated with tumor invasion (P = 0.0357), lymph nodes metastasis (P = 0.0272), a higher clinical stage (P = 0.0041), and poorer overall survival (P = 0.0105). Luciferase assays revealed that miR-520d-3p inhibited EphA2 expression by targeting the 3′-untranslated region of EphA2 mRNA. Overexpression of miR-520d-3p dramatically inhibited the proliferation, cell cycle progression, invasion, and migration of GC cells, while down-regulation substantially promoted these properties. Moreover, c-Myc, CyclinD1, and matrix metalloproteinase-9 expression levels were down-regulated in miR-520d-3p mimic-transfected cells and up-regulated in miR-520d-3p inhibitor-transfected cells. Taken together, our data showed that miR-520d-3p appears to contribute to GC progression via the regulation of EphA2 and could serve as a novel prognostic and potential therapeutic marker.     

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

Glycosylation and subsequent malonylation of isoflavonoids in E. coli: strain development,production and insights into future metabolic perspectives

Genistin and daidzein exhibit a protective effect on DNA damage and inhibit cell proliferation. Glycosylation and malonylation of the compounds increase water solubility and stability. Constructed pET15b-GmIF7GT and pET28a-GmIF7MAT were used for the transformation of Escherichia coli and bioconversion of genistein and daidzein. To increase the availability of malonyl-CoA, a critical precursor of GmIF7MAT, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinia were also introduced. Thus, the isoflavonoids were glycosylated at position 7 by 7-O-glycosyltranferase and were further malonylated at position 6^″ of glucose by malonyl-CoA: isoflavone 7-O-glucoside-6^″-O-malonyltransferase both from Glycine max. Engineered E. coli produced 175.7 µM (75.90 mg/L) of genistin and 14.2 µM (7.37 mg/L) genistin 6″-O-malonate. Similar conditions produced 162.2 µM (67.65 mg/L) daidzin and 12.4 µM (6.23 mg/L) daidzin 6″-O-malonate when 200 µM of each substrate was supplemented in the culture. Based on our findings, we speculate that isoflavonoids and their glycosides may prove useful as anticancer drugs with added advantage of increased solubility, stability and bioavailability.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

Production of eicosapentaenoic acid (EPA, 20:5n-3) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) through the alternative ∆8 desaturation pathway mediated by particle bombardment

Very long chain polyunsaturated fatty acids (VLCPUFAs), especially eicosapentaenoic acid (EPA, 20:5?^5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6?^{4,7,10,13,16,19}) have demonstrated important roles in a number of aspects of human health. Currently, our primary dietary sources for these fatty acids are from marine fish. Producing VLCPUFAs in oilseed crop by metabolic engineering was considered to provide an alternative, sustainable sources. Here, three heterologous genes, ?9 elongase (?9-Elo) of Isochrysis galbana, ?8 desaturase (?8-Des) of Euglena gracilis, ?5 desaturase (?5-Des) of Mortierella alpina, were co-transformed into maize inbred line Qi319 using a particle bombardment transformation method to produce EPA through the alternative ?8 desaturation synthetic pathway. A total of 144 herbicide resistant lines were obtained with an average transformation efficiency of 7.95%, of which 98 lines contain the Bar genes with the positive transformation efficiency of 4.74%. In addition, 60 of the 98 positive transgenic lines were identified to contain all three transgenes, ?9-Elo, ?8-Des, and ?5-Des. The fatty acid composition of the leaves from the 60 transgenic lines were subjected to gas liquid chromatography analysis and the results showed that the amounts of EPA reached 1.99% in an individual leaf. These data demonstrate the feasibility for the heterologous production of EPA in maize and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA, in maize by metabolic engineering in the future.     

Molecular typing and phenotype characterization of methicillin-resistant Staphylococcus aureus isolates from blood in Taiwan

Background

Staphylococcus aureus causes a variety of severe infections such as bacteremia and sepsis. At present, 60–80% of S. aureus isolates from Taiwan are methicillin resistant (MRSA). It has been shown that certain MRSA clones circulate worldwide. The goals of this study were to identify MRSA clones in Taiwan and to correlate the molecular types of isolates with their phenotypes.

Methods

A total of 157 MRSA isolates from bacteremic patients were collected from nine medical centers. They were typed based on polymorphisms in agr, SCCmec, MLST, spa, and dru. Phenotypes characterized included Panton-Valentine leucocidin (pvl), inducible macrolide-lincosamide-streptogramin B resistance (MLSBi), vancomycin (VA) and daptomycin (DAP) minimal inhibitory concentrations (MIC), and superantigenic toxin gene profiles. Difference between two consecutive samples was determined by Mann-Whitney-U test, and difference between two categorical variables was determined by Fisher''s exact test.

Results

Four major MRSA clone complexes CC1, CC5, CC8, and CC59 were found, including 4 CC1, 9 CC5, 111 CC8, and 28 CC59 isolates. These clones had the following molecular types: CC1: SCCmecIV and ST573; CC5: SCCmecII and ST5; CC8: SCCmecIII, ST239, and ST241, and CC59: SCCmecIV, SCCmecV_T, ST59, and ST338. The toxin gene profiles of these clones were CC1: sec-seg-(sei)-sell-selm-(seln)-selo; CC5: sec-seg-sei-sell-selm-(seln)-selp-tst1; CC8: sea-selk-selq, and CC59: seb-selk-selq. Most isolates with SCCmecV_T, ST59, spat437, and dru11 types were pvl ⁺ (13 isolates), while multidrug resistance (≥4 antimicrobials) were associated with SCCmecIII, ST239, spa t037, agrI, and dru14 (119 isolates) (p<0.001). One hundred and twenty four isolates with the following molecular types had higher VA MIC: SCCmecII and SCCmecIII; ST5, ST239, and ST241; spa t002, t037, and t421; dru4, dru10, dru12, dru13, and dru14 (p<0.05). No particular molecular types were found to be associated with MLSBi phenotype.

Conclusions

Four major MRSA clone complexes were found in Taiwan. Further studies are needed to delineate the evolution of MRSA isolates.     

Cryostorage of cloned amino Acid analog-resistant carrot and tobacco suspension cultures

Five clones were isolated from five different amino acid analog-resistant Daucus carota L. var. Sativa and Nicotiana tabacum L. cv. Xanthi cell lines. The individual clones were similar in their resistance to dl-5-methyltryptophan, S-(2-aminoethyl)-l-cysteine, or azetidine-2-carboxylic acid, and in their corresponding free amino acid levels.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.     

Linkage of the Salmonella typhimurium chromosomal loci encoding for the cytochrome-linked l-α-glycerophosphate dehydrogenase and amylomaltase activities

Mutant SM6, of Glp^- Mal^- phenotype, was isolated from Salmonella typhimurium LT2 after treatment with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant was able to use neither glycerol nor l-α-glycerophosphate, nor maltose as carbon source. Enzyme and transport assays indicated that glycerol kinase and l-α-glycerophosphate permease were present. No activity of the cytochrome linked l-α-glycerophosphate dehydrogenase could be demonstrated. This activity was present in LT2 cells grown in the presence of glycerol. These results were interpreted as a glpD^- character in the mutant. The inability to utilize maltose as carbon source was due to lack of the amylomaltase activity. The second enzyme needed for the utilization of maltose, maltodextrine phosphorylase, was present in mutant SM6 at normal levels. The mutant was thus malQ^-. By conjugation, both affected loci were located at about minutes 111–112 on the bacterial chromosome. By transduction it was found that there was approximately a 20% linkage between the two affected loci.     

The effects of galectin-1 on the gene expression of the transcription factors TBX21, GATA-3, FOXP3 and RORC

CD4⁺ T cells orchestrate the immune response by differentiating into T helper (Th) or regulatory (Treg) cell subsets that secrete distinct sets of cytokines. They also play a critical role in the pathogenesis of autoimmunity, asthma, allergy and, likely, cancer. The mechanisms involved in the regulation of CD4⁺ T cell homeostasis by galectin-1 remain poorly characterized. To investigate whether galectin-1 modulates the differentiation of CD4⁺ T cells, the effects of galectin-1 on the mRNA expression levels of TBX21, GATA-3, FOXP3 and RORC in activated peripheral blood mononuclear cells were examined. The expression levels of GATA-3 and FOXP3 mRNA were up-regulated after treatment with 1.0 μg/ml galectin-1 and were unchanged (for GATA-3) or slightly elevated (for FOXP3) compared with untreated cells when 2.0 μg/ml galectin-1 was added. At the same time, at both concentrations of galectin-1, we observed reduced TBX21 and RORC mRNA expression levels. These findings support the concept that galectin-1 skews the differentiation of CD4⁺ T cells towards Th2 and Treg cells.     

Synthesis and characterization of iodopropyl derivatives of adenosine 3′,5′-cyclic-monophosphate: Potential affinity labels of cAMP binding sites in enzymes

The syntheses of two potential cAMP affinity lables, 1,N ⁶-(3-iodopropyleno)adenosine 3′,5′-cyclic-monophosphate and 2′-O-(2-iodo-3-hydroxypropyl) adenosine 3′,5′-cyclic-monophosphate, by a two-step chemical procedure are described. TheN ⁶- and 2′-O-allyl intermediates were prepared selectively by alkylation of cAMP in organic and alkaline aqueous solutions, respectively. Treatment of theN ⁶-allyl derivative withN-iodosuccinimide resulted in iodine addition to the double bond and cyclization to theN ¹ position of the purine ring. The iodohydrin analog was synthesized by reaction of 2′-O-allyl-cAMP with potassium iodide and thallium trichloride in acetate buffered solution. The products were isolated by column chromatography and characterized by thin-layer chromatography, elemental analysis, and ultraviolet,¹³C, and¹H NMR spectroscopy. The cAMP analogs were found to react with lysine and cysteine. Both cAMP derivatives were tested for their reaction with the low-K _m cAMP phosphodiesterase of human platelets. The ribose-substituted analog functioned as a competitive inhibitor (K _I =0.72 μM) and caused a time-dependent irreversible inactivation of the phosphodiesterase. In contrast, the purine-substituted derivative acted neither as a reversible competitive inhibitor nor as an irreversible inactivator of the enzyme. These results indicate the specificity of these potential cAMP analogs in their interaction with the phosphodiesterase.     

l-phenylalanine production by double auxotrophic multianalogue-resistant mutant ofArthrobacter globiformis

Tryptophan-plus-tyrosine double auxotrophic mutants resistant to fluorophenylalanine (PFP) and β-2-thienylalanine (TA) were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in mineral salts medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to 5-methyltryptophan (MT) and 3-nitrotyrosine (NT) from a double auxotrophic PFP^r and TA^r mutant. Under optimal cultural condition one mutant yielded 9.6g phenylalanine per L medium in flask culture. Enzymic activity of regulatory enzymes (deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydratase) were observed in the wild type, double auxotroph and double-auxotrophic multianalogue-resistant mutant.     

9-O-acetylated sialic acids differentiating normal haematopoietic precursors from leukemic stem cells with high aldehyde dehydrogenase activity in children with acute lymphoblastic leukaemia

Childhood acute lymphoblastic leukaemia (ALL) originates from mutations in haematopoietic progenitor cells (HPCs). For high-risk patients, treated with intensified post-remission chemotherapy, haematopoietic stem cell (HSC) transplantation is considered. Autologous HSC transplantation needs improvisation till date. Previous studies established enhanced disease-associated expression of 9-O-acetylated sialoglycoproteins (Neu5,9Ac₂-GPs) on lymphoblasts of these patients at diagnosis, followed by its decrease with clinical remission and reappearance with relapse. Based on this differential expression of Neu5,9Ac₂-GPs, identification of a normal HPC population was targeted from patients at diagnosis. This study identifies two distinct haematopoietic progenitor populations from bone marrow of diagnostic ALL patients, exploring the differential expression of Neu5,9Ac₂-GPs with stem cell (CD34, CD90, CD117, CD133), haematopoietic (CD45), lineage-commitment (CD38) antigens and cytosolic aldehyde dehydrogenase (ALDH). Normal haematopoietic progenitor cells (ALDH⁺SSC^loCD45^hiNeu5,9Ac₂ -GPs^loCD34⁺CD38^?CD90⁺CD117⁺CD133⁺) differentiated into morphologically different, lineage-specific colonies, being crucial for autologous HSC transplantation while leukemic stem cells (ALDH⁺SSC^loCD45^loNeu5,9Ac₂ -GPs^hiCD34⁺CD38⁺CD90^?CD117^?CD133^?) lacking this ability can be potential targets for minimal residual disease detection and drug-targeted immunotherapy.