Optimization of a <Emphasis Type="Italic">Bacopa monnieri</Emphasis>-based genetic transformation model for testing the expression efficiency of pathway gene constructs of medicinal crops

A fast regenerating Agrobacterium tumefaciens-mediated transformation protocol for Bacopa monnieri (L.) Wettst. was developed as a model system for heterologous expression of terpenoid indole alkaloid pathway genes from Catharanthus roseus (L.) G. Don. The direct regeneration of shoots from leaf explants co-cultured with A. tumefaciens resulted in the integration of a tryptophan decarboxylase (tdc) and strictosidine synthase (str) cassette (<hpt-<Tdc2-<Str-gus>) in the regenerated progeny. The highest transformation efficiency (83.88%) was achieved when leaf explants were infected on the adaxial laminar surface by manual pricking with 48- to 72-h-old suspensions (OD₆₀₀ = 0.5–0.6) of A. tumefaciens strain LBA1119 (carrying the binary vector pMOG22). The heterologous expression of tryptophan decarboxylase and strictosidine synthase genes that are otherwise not present in B. monnieri plants was confirmed through semi-quantitative PCR and metabolite quantification assays. The entire protocol duration from co-cultivation through regeneration of transgenic plants to their establishment in the glass house took 40–45 d. The developed B. monnieri model can be used to test expression cassettes carrying genes for plant secondary metabolic pathway engineering, especially those genes that are expressed in differentiated cell, tissue, or organs.     

Natural polyphenolic inhibitors against the antiapoptotic BCL-2

The apoptotic mechanism is regulated by the BCL-2 family of proteins, such as BCL-2 or Bcl-xL, which block apoptosis while Bad, Bak, Bax, Bid, Bim or Hrk induce apoptosis. The overexpression of BCL-2 was found to be related to the progression of cancer and also providing resistance towards chemotherapeutic treatments. In the present study, we found that all polyphenols (apigenin, fisetin, galangin and luteolin) bind to the hydrophobic groove of BCL-2 and the interaction is stable throughout MD simulation run. Luteolin was found to bind with highest negative binding energy and thus, claimed highest potency towards BCL-2 inhibition followed by fisetin. The hydrophobic interactions were found to be critical for stable complex formation as revealed by the vdW energy and ligplot analysis. Finally, on the basis of data obtained during the study, it can be concluded that these polyphenols have the potential to be used as lead molecules for BCL-2 inhibition.     

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4. The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas, land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.     

Cytomorphological spectrum in gynaecomastia: a study of 389 cases

Cytomorphological spectrum in gynaecomastia: a study of 389 cases The objective of this study was to document the spectrum of cytomorphological features in aspirates from gynaecomastia. During a 22-year period (July 1979 to June 2001) fine needle aspirations (FNA) were conducted on 651 males with a breast lesion. Fine needle aspirate slides from 529 benign cases were reviewed along with the histological sections of 54 cases where available. Of the 529 FNA, 140 were excluded (105 unsatisfactory, five cystic fluid aspirated, 30 inflammatory lesions). In 134 of 389 cases reviewed florid gynaecomastia was observed while the remainder showed benign ductal cells (BDC) in varying quantities from less than five to more than 25 groups. Of the 134 cases with florid gynaecomastia, 49 had a fibroadenoma-like picture (FLP), where a large number of BDC fragments with a staghorn configuration, prominent stromal fragments with contoured stroma and bare bipolar nuclei were seen abundantly in all cases. In 85 cases the features resembled fibroadenomatoid hyperplasia (FH), with only an occasional staghorn configuration of BDC seen in 82% of the cases. In 83% of cases stromal fragments of varying proportions were seen but a contoured stroma was seen in only 3.5% of cases. Both stromal and epithelial atypia were seen in 41% and 86% cases, respectively, of FLP, and in 7% and 69% cases of FH. Columnar cells, apocrine cells and foam cells were seen in approximately similar quantities in both the lesions. Vascularized stroma was seen in 22% of the cases with FLP but in only 4.5% of cases those with FH. Cytohistological correlation performed in 54 cases showed marked variation. Fine needle aspirates showed florid changes in 13 of the 41 cases of simple gynaecomastia and two of the eight cases with florid gynaecomastia on tissue sections. In conclusion, a wide spectrum of morphological features are encountered in FNA from benign male breast lesions. It is important to recognize these to avoid a false-positive diagnosis.     

Activation of the p38 mitogen-activated protein kinase mediates the suppressive effects of type I interferons and transforming growth factor-beta on normal hematopoiesis.

Type I interferons (IFNs) are potent regulators of normal hematopoiesis in vitro and in vivo, but the mechanisms by which they suppress hematopoietic progenitor cell growth and differentiation are not known. In the present study we provide evidence that IFN alpha and IFN beta induce phosphorylation of the p38 mitogen-activated protein (Map) kinase in CD34+-derived primitive human hematopoietic progenitors. Such type I IFN-inducible phosphorylation of p38 results in activation of the catalytic domain of the kinase and sequential activation of the MAPK-activated protein kinase-2 (MapKapK-2 kinase), indicating the existence of a signaling cascade, activated downstream of p38 in hematopoietic progenitors. Our data indicate that activation of this signaling cascade by the type I IFN receptor is essential for the generation of the suppressive effects of type I IFNs on normal hematopoiesis. This is shown by studies demonstrating that pharmacological inhibitors of p38 reverse the growth inhibitory effects of IFN alpha and IFN beta on myeloid (colony-forming granulocytic-macrophage) and erythroid (burst-forming unit-erythroid) progenitor colony formation. In a similar manner, transforming growth factor beta, which also exhibits inhibitory effects on normal hematopoiesis, activates p38 and MapKapK-2 in human hematopoietic progenitors, whereas pharmacological inhibitors of p38 reverse its suppressive activities on both myeloid and erythroid colony formation. In further studies, we demonstrate that the primary mechanism by which the p38 Map kinase pathway mediates hematopoietic suppression is regulation of cell cycle progression and is unrelated to induction of apoptosis. Altogether, these findings establish that the p38 Map kinase pathway is a common effector for type I IFN and transforming growth factor beta signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis.     

Seasonal Changes in Fungal Production and Biomass on Standing Dead Scirpuslacustris Litter in a Northern Prairie Wetland

Decaying macrophytes are an important source of carbon and nutrients in fungal and bacterial communities of northern prairie wetlands. Dead macrophytes do not collapse into the water column immediately after death, and decomposition by fungi and bacteria begins while the plants are standing. The seasonal variations in fungal biomass and production on Scirpus lacustris stems, both above and below water, were measured to assess which environmental factors were dominant in affecting these variations in a typical prairie wetland. Fungal biomass and production were measured from early May to November, just prior to freeze-up. Fungal decomposition began and was greatest in the spring despite low water temperatures. The fungal production, as measured by the incorporation of [1-¹⁴C]acetate into ergosterol, ranged from 1.8 to 376 μg of C g of ash-free dry mass (AFDM)⁻¹ day⁻¹, and the biomass, as estimated by using ergosterol, ranged from nondetectable to 5.8 mg of C g of AFDM⁻¹. There was no significant difference in biomass or production between aerial and submerged portions of Scirpus stems. The water temperature was correlated with fungal production (r = 0.7, P < 0.005) for aerial stem pieces but not for submerged pieces. However, in laboratory experiments water temperature had a measurable effect on both biomass and production in submerged stem pieces. Changes in fungal biomass and productivity on freshly cut green Scirpus stems decaying in the water either exposed to natural solar radiation or protected from UV radiation were monitored over the summer. There was no significant difference in either fungal biomass (P = 0.76) or production (P = 0.96) between the two light treatments. The fungal biomass and rates of production were within the lower range of the values reported elsewhere, probably as a result of the colder climate and perhaps the lower lability of Scirpus stems compared to the labilities of the leaves and different macrophytes examined in other studies performed at lower latitudes.     

Esterase isozyme polymorphism, specific and nonspecific esterase, syngenic lines development and natural occurrence of a thermostable esterase in the tropical silkworm Bombyx mori L.

Esterase isozyme polymorphism was documented for digestive juice and haemolymph of the tropical multivoltine silkworm, Bombyx mori L., breed CB5 (GP) and its syngenic lines (CB5Lm^e-1, CB5Lm-2 and CB5Lm-5) using α- and β-naphthylacetate separately as nonspecific substrates (Ogita, Z., Kasai, T., 1965. Genetico-biochemical analysis of specific esterases in Musca domestica. Jpn. J. Genet. 40, 173–184). Polymorphism existed in the isozyme pattern of α-esterase with two or three bands in digestive juice and three to five bands in haemolymph. No polymorphism was observed in β-esterase isozyme pattern having four bands in digestive juice and two bands in haemolymph. During the course of esterase isozyme studies, the presence of some specific α-esterase bands (Est-1, 4 and 5) in haemolymph and β-esterase bands (Est-1, 2 and 3) in digestive juice were observed. But both α- and β-esterase bands Est-3 and 4 in digestive juice and Est-2 and 3 in haemolymph were found to be nonspecific. Nonspecific β-esterase band (Est-3) in haemolymph of CB5 (GP) and its syngenic lines withstood a temperature up to 80±1°C for 10 min. No thermostable band was observed in the isozyme zymogram of α-esterase in digestive juice and haemolymph or β-esterase in digestive juice. Overall, this study discusses the presence of esterase heterogeneity in the CB5 (GP) genepool, syngenic lines development, occurrence of specific α- and β-esterase bands in digestive juice and haemolymph and thermostable β-esterase band Est-3 in haemolymph in tropical silkworm Bombyx mori L.     

Seasonal variation in methane emission from a temperate Phragmites-dominated marsh: effect of growth stage and plant-mediated transport

The eddy covariance technique was employed with a tunable diode laser spectrometer to quantify methane flux from a prairie marsh dominated by Phragmites australis in north-central Nebraska, USA. The observations spanned the entire growing season (April to October) and a wide range of weather conditions, allowing a quantitative assessment of the physical and biological controls of methane emission in this ecosystem. Diel patterns in methane emission varied markedly depending on plant growth stage. Prior to plant emergence above water, the rate of methane emission from the marsh was fairly constant throughout the day. After emergence above water, there was a gradual increase in methane emission after sunrise with a peak in late afternoon. Significant changes in diel patterns were observed after tillering. Then, the diel pattern was characterized by a mid- to late-morning peak and a 2-to 4-fold increase in methane emissions from night to daytime. In early stages of plant growth, molecular diffusion through dead/live plants and the standing water column seemed to be the primary transport mechanism. After tillering, a transition occurred in the transport mechanism from a molecular diffusion to a convective throughflow, which is a rapid and active gas transport driven by pressure differences. The role of convective throughflow became less important as the plants senesced. Integrated methane emission over the six-month measurement period (April–October) was about 64 g CH₄ m^–2. On an annual basis, we estimate the annual methane emission from this ecosystem to be ≈ 80 g CH₄ m^–2 and that about 80% of the total methane emission occurred between late April and late October.