scholarly journals Basic β-1,3-Glucanase from Drosera binata Exhibits Antifungal Potential in Transgenic Tobacco Plants

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1747
Author(s):  
Miroslav Rajninec ◽  
Monika Fratrikova ◽  
Eva Boszoradova ◽  
Martin Jopcik ◽  
Miroslav Bauer ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Crude Protein ◽  
Trichoderma Viride ◽  
Alternaria Solani ◽  
Fold Increase ◽  
Transcript Abundance ◽  
Carnivorous Plant ◽  
Vitro Assay ◽  
Antifungal Efficiency

The basic β-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total β-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified β-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14–34%). Further analyses revealed that the complementary action of transgenic β-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

scholarly journals Effect of Inoculations of Trichoderma viride and Saccharomyces cerevisiae Mixed Culture on Chemical Composition, Fiber, Digestibility and Theobromine Cocoa Pod Fermentation

2016 ◽  
Vol 4 (2) ◽  
pp. 178 ◽  
Author(s):  
Muhammad Askari Zakariah
Keyword(s):  
Chemical Composition ◽  
Mixed Culture ◽  
Crude Protein ◽  
Trichoderma Viride ◽  
In Vitro Digestibility ◽  
Chemical Compositions ◽  
Fiber Fraction ◽  
Nitrogen Free Extract ◽  
Significant Difference

The objective of the study was to identify the effect of Trichoderma viride andSaccharomyces cerevisiae inoculant on chemical compositions, digestibility, and theobromineconcentration of fermented cocoa pod. This experiment consisted of four treatments,namely cocoa pods without fermentation as control (R0); fermentation of cocoa pods withinoculant T. viride (R1); fermentation of cocoa pods with inoculant S. cerevisiae (R2); andfermentation of cocoa pods with inoculant T. viride and S. cerevisiae mixed culture (R3).Each treatment had 3 replicates, and then was fermented for 10 days. Variables observedwere the chemical compositions i.e dry matter (DM), organic matter (OM), crude protein(CP), ether extract (EE), crude fiber (CF), nitrogen free extract (NFE), fiber fraction (Neutraldetergent fiber and acid detergent fiber), in vitro digestibility, and theobromine concentration.Data were analysed by one-way analysis of variance and followed by Duncan’s new multiplerange test (DMRT), if there were any significant difference. Results showed the inoculumaffected (P<0.05) the chemical composition, fiber fraction and in vitro digestibility. However,theobromine was not detected on cocoa pod without fermentation and fermentation.Compared to group R0, inoculation with T. viride and S. cerevisiae mixed culture (P<0.05)resulted in higher DM concentration (92.78% vs 89.72% respectively), higher CP (7.43% vs5.63% respectively), higher NDF (79.41% vs 61.18% respectively), higher ADF (73.04%vs 47.94% respectively), but was not significantly different on DM and OM digestibility(21.22% vs 22.24%, and 22.67% vs 24.31% respectively) than cocoa pod without fermentation.It is concluded that inoculant T. viride and S. cerevisiae mixed culture increased CPconcentration, but had no effect on in vitro digestibility.

scholarly journals PROTEIN FRACTIONATION AND UTILIZATION OF SOYBEAN AND REDBEAN AS AFFECTED BY DIFFERENT DRYING TEMPERATURE

2017 ◽  
Vol 41 (1) ◽  
pp. 37
Author(s):  
Anuraga Jayanegara ◽  
Yesi Chwenta Sari ◽  
Roni Ridwan ◽  
Didid Diapari ◽  
Erika Budiarti Laconi
Keyword(s):  
Chemical Composition ◽  
Crude Protein ◽  
Volatile Fatty Acids ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Vitro Assay ◽  
Drying Temperature ◽  
Higher Temperature ◽  
In Vitro Rumen Fermentation

The objective of this study was to investigate the influence of different drying temperature on chemical composition, in vitro rumen fermentation and digestibility of soybean and redbean. Soybean and redbean were dried in an oven set at four different drying temperatures, i.e. 50, 60, 70 and 80 oC for 24 h in three replicates. Dried samples were then milled and used further for chemical composition determination (proximate analysis, Van Soest analysis and protein fraction) and in vitro rumen fermentation assay. Parameters measured in the in vitro assay were gas production, digestibility, pH, ammonia and volatile fatty acids (VFA). Data obtained were analyzed by using analysis of variance and a posthoc test namely Duncan’s multiple range test. Results revealed that neutral detergent insoluble crude protein (NDICP) content increased at higher drying temperature (70 or 80 oC) for both soybean and redbean (P<0.05) but at different magnitude. As with NDICP, higher temperature led to a higher acid detergent insoluble crude protein (ADICP) both in soybean and redbean (P<0.05). Higher temperature decreased gas production rate (GPR) of both beans (P<0.05). Drying of soybean at 70 or 80 oC decreased crude protein digestibility (CPD) of soybean than dried at 50 or 60 oC (P<0.05). Higher drying temperature resulted in a lower NH3 concentration (P<0.05). It can be concluded that drying temperature at 50 or 60 oC is safe to maintain nutritional quality of soybean and redbean.

scholarly journals Control of Early Blight of Tomato Caused by Alternaria Solani and Screening of Tomato Varieties against the Pathogen

2019 ◽  
Vol 13 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Chapol K. Roy ◽  
Nafiza Akter ◽  
Mohammad K.I. Sarkar ◽  
Moyen Uddin Pk ◽  
Nadira Begum ◽  
...  
Keyword(s):  
Azadirachta Indica ◽  
Early Blight ◽  
Allium Sativum ◽  
Trichoderma Viride ◽  
Reduction Process ◽  
Alternaria Solani ◽  
Disease Index ◽  
Common Disease ◽  
Dual Culture

Introduction:Early blight is a common disease of tomato, which is caused byAlternaria solani.Objectives:This work was accompanied to find an alternative to chemical fungicides and to screen tomato varieties againstAlternaria solani.Methods:The infected leaves were collected from five tomato fields of Shere-e-Bangla Agricultural University, Dhaka and were cultured for the identification of the infectious fungus and The phytobiocidal role of six plants againstAlternaria solaniwas evaluatedin vitromodel.Results & Discussion:Alternaria solaniwas identified as the infectious fungus. The growth of the test fungiTrichodermaspp.viz.,Trichoderma viride,T. harzianumcollected form NAMDEC andTrichodermasp collected from field of BCSIR was monitored as optimum PH. All the selectedTrichodermaspp. were antagonistic toA. solani.Antagonistic capacity of theTrichodermaspp. was tested by dual culture, volatile as well as non-volatile method. It was observed,T. viridewas most effective in the reduction process ofA. solaniandT. harzianum.T.viridealso showed highest inhibition in volatile and non-volatile trials. Six plant extractsviz.,Adhatoda vasica(Nees),Azadirachta indica(A Juss).Ocimujm sanctum(L),Allium sativum(L),Datura metal(Linn) andZingiber officinale(Rose) were selected to evaluate theirin vitroefficacy of 5%, 10% and 20% concentration against theA. solani.Allium sativumwas the most effective one againstA. solani, followed byAzadirachta indica. The efficacy of five fungicidesviz., Bavistin 50WP, Mancozeb 80WP, Indofil M-45, Sulcox 50WP and Tall 25EC were evaluated for their fungitoxicity against theA. solaniat 100, 200,100, 600 and 800 ppm. Tall 25EC was the most effective fungicide againstAlternaria solanifollowed by Mancozeb 80WP. After screening the five tomato varieties againstA. solani, it was revealed that BARI Tomato-9 had the highest Percentage of Disease Index (PDI) and the leaf of BARI Tomato-7 had the lowest Percentage of Disease Index (PDI).Conclusion:The extract ofAllium sativumwas effective to controlAlternaria solaniat prescribed concentration. The highest PDI was found in BARI tomato-9 againstAlternaria solani.

Abstract 580: Acidic Domain of ApoB-100 and Other Short Negatively Charged Peptides Anchored to Lipoproteins Cause Hypertriglyceridemia in Mice

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Denis O Sviridov ◽  
Puja Terse ◽  
Anna Wolska ◽  
Scott M Gordon ◽  
Alan T Remaley
Keyword(s):  
Amino Acid ◽  
Fold Increase ◽  
Alpha Tocopherol ◽  
Binding Motif ◽  
Vitro Assay ◽  
Acidic Domain ◽  
Ldl Particles ◽  
Control Peptide ◽  
Charged Peptides

Human Apolipoprotein B-100 (ApoB-100) is the main protein of VLDL and LDL particles in plasma. It is a large 4536 amino acid protein with various structural domains, such as the LDL-receptor binding motif and multiple proteoglycan binding sites, all of which have a high positive charge. A search of the primary amino acid sequence of ApoB-100 revealed the presence of the following acidic domain: DMDEDDD (AA 3985-3991). We hypothesized that this motif could affect the interaction of ApoB-containing lipoproteins with liver and or possibly GPIHBP1, which also contains an acidic domain that interacts with the positive charged motifs on VLDL. We synthesized a fluorescent tagged DMDEDDD peptide and conjugated it at the N-terminus with two molecules of alpha-tocopherol to serve as an anchor to lipoprotein particles. After adding the peptide to human plasma, it was found to readily bind to all lipoproteins, as determined by FPLC analysis. VLDL incubated with the peptide showed an increased negative charge and lost its ability to bind to heparin as determined by chromatography on a HiTrap TM heparin column. The peptide injected in LDLr-KO mice (30 mg/kg) caused a rapid 3-fold increase in plasma triglycerides within 1 h and the triglyceride elevation persisted for at least 24 h. A similarly designed negative charged peptide attached to tocopherol (EEEEEEEE) also raised triglycerides in mice but a neutral charged control peptide (KEKEKEKE) did not. To understand the mechanism, we tested the above peptides for their ability to inhibit LPL, using an in vitro assay with intralipid as a substrate and found that negative charged peptides inhibited lipolysis (IC 50= 50 uM), but the neutral control peptide did not. We are now investigating other potential mechanisms for how acidic peptides may cause hypertriglyceridemia. In summary, a peptide based on an acidic domain on ApoB-100 appears to cause hypertriglyceridemia in mice by inhibition of LPL and possibly by interfering with proteoglycan binding of lipoproteins. These results may be relevant in understanding VLDL metabolism and for the design of therapeutic apolipoprotein mimetic peptides.

Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications

2018 ◽  
Vol 73 (3-4) ◽  
pp. 95-105 ◽  
Author(s):  
Mohamed K. Al-Essa ◽  
Susanne Melzer ◽  
Attila Tarnok ◽  
Kamal A. Hadidi ◽  
Mohammed El-Khateeb
Keyword(s):  
Succinic Anhydride ◽  
Fluorescent Dyes ◽  
Fluorescein Isothiocyanate ◽  
Fold Increase ◽  
Fluorescent Dye ◽  
Cooh Group ◽  
Vitro Assay ◽  
Bioanalytical Applications ◽  
Ethanesulfonic Acid

AbstractThis study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4′,6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Abstract 293: Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Vasculogenesis.

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Lucinda Low ◽  
Christine Cheung ◽  
Martin R Bennett ◽  
Sanjay Sinha
Keyword(s):  
Network Formation ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Confocal Imaging ◽  
Paraxial Mesoderm ◽  
Hesc Line ◽  
Vitro Assay ◽  
Matrigel Assay ◽  
Network Area

Lineage-tracking studies in avian and mouse embryos have revealed that SMCs in different regions of the vasculature are derived from different embryological origins. We have developed an in vitro protocol to differentiate human embryonic stem cells (hESCs) into SMCs through different embryonic lineages, namely neuroectoderm, lateral plate mesoderm (LM) and paraxial mesoderm. As mural cells are thought to augment the formation of functional blood vessels during revascularisation, this study aims to determine whether embryologically-distinct SMCs differ in their ability to support vasculogenesis. We assessed the hypothesis that LM-SMCs are superior in supporting endothelial network formation in vitro. Using an mStrawberry-expressing hESC line, we derived the three origin-specific SMCs and co-cultured them with GFP-expressing HUVECs in a 3D in vitro matrigel assay. Endothelial network formation was assessed using real-time confocal imaging. Quantitative analysis revealed that LM-SMCs alone had a supportive effect on network formation and survival, with an increase in HUVEC network area after 4 days (3.23-fold increase ± 1.04-fold vs HUVECs alone; p<0.05; n=3) and 8 days (6.38-fold increase ± 0.39-fold vs HUVECs alone; p<0.001; n=3). In addition, LM-SMCs appear to facilitate more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. To identify whether the LM-SMC-specific influence on network formation and survival was a paracrine effect, the three SMC types were tested in a paracrine 3D in vitro assay, where they shared media with HUVECs seeded in an adjacent well. Preliminary results reveal that the supportive effect of LM-SMCs is, at least partly, paracrine, with an increase in HUVEC network area after 4 days (7.08-fold increase vs HUVECs alone; n=1) and 8 days (21.39-fold increase vs HUVECs alone; n=1). Currently, we are exploring possible soluble factors that may be responsible and investigating this effect in an in vivo matrigel assay. In conclusion, the embryological origin of SMCs influences their functional ability to support vasculogenesis. This research will provide insight into which SMC type will be most effective in future revascularisation therapy.

Src and phosphatidylinositol 3–kinase mediate soluble E-selectin–induced angiogenesis

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3960-3968 ◽  
Author(s):  
Pawan Kumar ◽  
Mohammad A. Amin ◽  
Lisa A. Harlow ◽  
Peter J. Polverini ◽  
Alisa E. Koch
Keyword(s):  
Fold Increase ◽  
Tube Formation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Microvascular Endothelial Cell ◽  
Vitro Assay ◽  
Pi3k Inhibitor Ly294002 ◽  
Soluble E Selectin

Abstract Angiogenesis plays an important role in a variety of pathophysiologic processes, including tumor growth and rheumatoid arthritis. We have previously shown that soluble E-selectin (sE-selectin) is an important angiogenic mediator. However, the mechanism by which sE-selectin mediates angiogenesis is still unknown. In this study, we show that sE-selectin is a potent mediator of human dermal microvascular endothelial cell (HMVEC) chemotaxis, which is predominantly mediated through the Src and the phosphatidylinositiol 3–kinase (PI3K) pathways. Further, sE-selectin induced a 2.2-fold increase in HMVEC tube formation in the Matrigel in vitro assay. HMVECs pretreated with the Src inhibitor (PP2) and the PI3K inhibitor (LY294002) or transfected with Src antisense oligonucleotides or Akt dominant-negative mutants significantly inhibited sE-selectin–mediated HMVEC tube formation. In contrast, HMVECs transfected with an extracellular signal-related kinase 1/2 (ERK1/2) mutant or pretreated with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 failed to show sE-selectin–mediated HMVEC tube formation. Similarly, in the Matrigel-plug in vivo assay, sE-selectin induced a 2.2-fold increase in blood vessel formation, which was significantly inhibited by PP2 and LY294002 but not by PD98059. sE-selectin induced a marked increase in Src, ERK1/2, and PI3K phosphorylation. PI3K and ERK1/2 phosphorylation was significantly inhibited by PP2, thereby suggesting that both of these pathways may be activated via Src kinase. Even though the ERK1/2 pathway was activated by sE-selectin in HMVECs, it seems not to be essential for sE-selectin–mediated angiogenesis. Taken together, our data clearly show that sE-selectin–induced angiogenesis is predominantly mediated through the Src-PI3K pathway.

Amino acid digestibility of meat and bone meals for broiler chickens

2002 ◽  
Vol 53 (11) ◽  
pp. 1257 ◽  
Author(s):  
V. Ravindran ◽  
W. H. Hendriks ◽  
B. J. Camden ◽  
D. V. Thomas ◽  
P. C. H. Morel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Crude Protein ◽  
Ash Content ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Vitro Assay ◽  
Ileal Digestibility ◽  
Gross Energy

Variation in the apparent ileal digestibility of amino acids in 19 meat and bone meal samples, obtained from commercial rendering plants in New Zealand, was determined using 5-week-old broilers. Assay diets contained meat and bone meal as the only source of protein, and chromic oxide as an indigestible marker for the calculation of amino acid digestibility values. Correlations of chemical composition (crude protein, ash, crude fat, and gross energy) and in vitro assays (protein solubility in 0.2% potassium hydroxide and nitrogen digestibility by 0.2% pepsin hydrolysis) with ileal amino acid digestibility were also examined. Considerable variation was observed in the contents of crude protein (38.5–67.2 g/100 g), ash (13.0– 56.5 g/100 g), crude fat (4.3–15.3 g/ 100�g), and gross energy (9.4–22.3 MJ/kg) of meat and bone meal samples. The amino acid concentrations and ileal digestibility of amino acids also varied substantially. Cystine, the first limiting amino acid in meat and bone meal, had the lowest digestibility estimates. Correlation analyses showed that the ash content was the only chemical parameter that was consistently correlated with amino acid digestibility. Digestibility of amino acids, with the exception of aspartic acid, threonine, serine, tyrosine, histidine, and cystine, was negatively correlated with ash content, with samples with high ash levels having lower digestibility. Both in vitro assay measurements were found to be insensitive indicators of variations in amino acid digestibility.

A Simple and Inexpensive Clinical Whole Body Counter

1976 ◽  
Vol 15 (05) ◽  
pp. 248-253
Author(s):  
A. K. Basu ◽  
S. K. Guha ◽  
B. N. Tandon ◽  
M. M. Gupta ◽  
M. ML. Rehani
Keyword(s):  
The Body ◽  
Whole Body ◽  
Vitro Assay ◽  
Body Counter ◽  
Optimum Parameters ◽  
Whole Body Counter ◽  
Single Detector ◽  
Background Index ◽  
Iodide Crystal

SummaryThe conventional radioisotope scanner has been used as a whole body counter. The background index of the system is 10.9 counts per minute per ml of sodium iodide crystal. The sensitivity and derived sensitivity parameters have been evaluated and found to be suitable for clinical studies. The optimum parameters for a single detector at two positions above the lying subject have been obtained. It has been found that for the case of 131I measurement it is possible to assay a source located at any point in the body with coefficient of variation less than 5%. To add to the versatility, a fixed geometry for in-vitro counting of large samples has been obtained. The retention values obtained by the whole body counter have been found to correlate with those obtained by in-vitro assay of urine and stool after intravenous administration of 51Cr-albumin.

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