Green Fluorescent Spots Research Articles

The dynamic change in the degradation and in vitro digestive properties of porcine myofibrillar protein during freezing storage

The myofibrillar protein (MP) degradation and in vitro digestive properties of porcine longissimus during freezing at −8, −18, −25 and − 40 °C for 1, 3, 6, 9 and 12 months were investigated. As the freezing temperature and duration of frozen storage increased, the amino nitrogen and TCA (trichloroacetic acid)-soluble peptides of the samples were significantly increased, while the total sulfhydryl content and band intensity of myosin heavy chain, actin, troponin T, tropomyosin were significantly decreased (P < 0.05). At higher freezing storage temperatures and durations, the particle size of MP samples and the green fluorescent spots detected using a laser particle size analyzer and confocal laser scanning microscopy became large. After 12 months of freezing, the digestibility and the degree of hydrolysis of the trypsin digestion solution of the samples frozen at −8 °C were significantly decreased by 15.02 % and 14.28 %, respectively, when compared to fresh samples, whereas, the mean surface diameter (d3,2) and mean volume diameter (d4,3) were significantly increased by 14.97 % and 21.53 %, respectively. Therefore, frozen storage induced protein degradation and impaired the ability of digestion in the pork proteins. This phenomenon was more evident as the samples were frozen at high temperatures over a long storage period.

Exogenous plant hormones alleviate As stress by regulating antioxidant defense system in Oryza sativa L.

Plant hormones play essential roles in plant growth regulation and resistance to environmental pressure. A hydroponic experiment was conducted using Zhongjiazao 17 rice to explore the effects of exogenous plant hormones on antioxidant response and As accumulation in rice under As stress. Melatonin (MT), 2,4-epibrassinolide (EBL), and jasmonic acid (JA) reduced the As content in seedlings significantly by 13.4% (MT)-32.5% (EBL) under 5µM As stress. Three hormones increased superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and glutathione (GSH) content significantly (2.2%-82.9%) in 5µM As stress condition, whereas the levels of H2O2 and malondialdehyde (MDA) were reduced significantly (32.3%-78.1%). Plant hormone addition reduced the As content in seedlings significantly by 18.2% (JA)-33.3% (MT) under 25µM As stress. SOD, POD, and CAT activities and GSH content in seedlings increased significantly (5.6-90.4%) with three hormones addition in 25µM As stress, whereas the levels of H2O2, O2˙¯, and MDA reduced significantly (20.9-73.0%). Staining with 2',7'-dichlorodihydrofluorescein diacetate and nitroblue tetrazolium showed that green fluorescence and blue spots decreased gradually in hormone-treated seedlings, further confirming that the exogenous addition of hormones weakened the oxidative stress of As to seedlings. Oxidative damage by As stress was reduced more by EBL than by the other hormones MT or JA. Totally, exogenous plant hormone can alleviate As stress in rice by activating enzyme activity of antioxidant defense system and scavenging reactive oxygen species, thus reducing oxidative damage and As accumulation in rice seedlings.

GFP transient expression and silencing in Fragaria x ananassa

BACKGROUND: Stable transformation, transient expression, and post-transcriptional gene silencing (PTGS) are powerful methodologies that allow exploration of gene function. OBJECTIVE: We aimed to apply these methodologies to strawberry leaves. Methods: the binary vectors pBIN19-sgfp, pBICdsGFP and pBIN61-P19 were transferred into A. tumefaciens EHA105 supervirulent strain by electroporation. The sgfp gene silencing was carried out in stably transformed GFP (green fluorescent protein) F. x ananassa Duch. cultivar ‘Pájaro’ strawberry plants by agroinfiltration. GFP-fluorescence was observed using a stereomicroscope (507 nm). RESULTS: We attained a GFP transgenic F. x ananassa plant that expresses the functional protein in all the tissues during a complete and normal life cycle. In planta sgfp transient expression and silencing have also been achieved in F. x ananassa cv. ‘Pájaro’ leaves of wild type and GFP transgenic plants, respectively. Agrobacterium-mediated transient expression was visualized as high intensity green fluorescent spots as early as 7 days post-agroinfiltration (dpa), peaking between 10 and 14 dpa and persisting as long as 24 dpa. A knockdown GFP phenotype was achieved by silencing using a dsGFP hairpin. CONCLUSION: This work contributes significantly to the reverse genetics field in strawberry, might help to gain knowledge in the analysis of functional promoters and thereby allow protein expression and silencing of genes. This will help to develop resistant plants expressing plant defense elicitors or silencing pathogen receptors and/or negative regulators of plant defense.

Effect of licochalcone A on autophagy in renal cell carcinoma via PI3K/Akt/mTOR signaling pathway

To investigate the effect of licochalcone (LCA) on autophagy in renal cell carcinoma (RCC), and further determine whether the mechanism is correlated with the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway, RCC 786-O and 769-P were used as study subjects. MTT assay was used to examine cell proliferation. The abilities of migration and invasion were detected by Transwell. The autophagy was observed under the fluorescence microscope through acridine orange staining. Green fluorescence spots were observed in Ad-GFP-LC3 transfection experiment. The protein expression was detected by Western blot. MTT assay results showed a dose and time-dependent cytotoxicity in the two cell lines treated with LCA. LCA inhibited migration and invasion in 786-O and 769-P cells. LCA increased the expression levels of LC3-Ⅱ, beclin 1, Atg5, and down-regulated the expression of p62. In addition, LCA inhibited the PI3K/Akt/mTOR pathway. Furthermore, the inhibition of PI3K/Akt by LY294002 or that of mTOR by rapamycin augmented LCA-induced autophagy. The findings demonstrated that LCA inhibited the proliferation, migration, invasion, and induced autophagy by inactivating PI3K/Akt/mTOR signaling pathway in 786-O and 769-P cells.

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens: Transient Expression of sgfp in Rice Calluses of indica cv. Fatmawati and japonica cv. Nipponbare

Transient expression of synthetic green fluorescent protein (sgfp) mediated by Agrobacterium is rapid and useful approach for visual monitoring the genetic transformation event in transformed cells/tissues of examined genotype. The significant differences in transient expression of two genotypes (indica cv. Fatmawati and japonica cv. Nipponbare) were found with regard to the osmotic treatment (0.4 M mannitol), solid subcultured callus, and 10 min. air drying. While, no significant differences in sgfp expression were observed in two genotypes on without air-drying and 5 min. air-drying of calluses prior immersed in Agrobacterium suspension. Surprisingly, the sgfp expression could not be detected in suspension-subcultured callus of both cultivars. The highest sgfp expression was achieved in Nipponbare callus treated with 10 min. air drying. The level of green fluorescent spots was higher in Nipponbare than that in Fatmawati, however, plants regenerated from Fatmawati were considerably comparable with those of Nipponbare. Seventeen and 16 putative transgenic rice plants expressing sgfp transgene were obtained from Nipponbare and Fatmawati, respectively.

Apoptosis-inducing effect of three medicinal plants on oral cancer cells KB and ORL-48.

Brucea javanica, Azadirachta indica, and Typhonium flagelliforme are medicinal plants commonly used to treat conditions associated with tumour formation. This study aimed to determine the antiproliferative activity of these plants extracts on KB and ORL-48 oral cancer cell lines and to suggest their mode of cell death. The concentration producing 50% cell inhibition (IC50) was determined and the activity was examined under an inverted microscope. Immunohistochemistry fluorescent staining method (TUNEL) was performed to indicate the mechanism of cell death and the fragmented DNA band pattern produced was obtained for verification. Compared to Azadirachta sp. and Typhonium sp., the antiproliferative activity of Brucea sp. extract was the most potent on both KB and ORL-48 cells with IC50 of 24.37 ± 1.75 and 6.67 ± 1.15 µg/mL, respectively. Signs of cell attrition were observed 24 hr after treatment. Green fluorescent spots indicating cell death by apoptosis were observed in images of both cells following treatment with all the three extracts. DNA fragments harvested from Brucea-treated cells produced bands in a ladder pattern suggesting the apoptotic effect of the extract. It is thus concluded that Brucea sp. extract exhibited cytotoxic activity on ORL-48 cells and their action mechanism is via apoptosis.

Comparison of immune responses against FMD by a DNA vaccine encoding the FMDV/O/IRN/2007 VP1 gene and the conventional inactivated vaccine in an animal model

Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP(1) gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1(+)-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together, conventional vaccine, PBS (as negative control), pcDNA3.1(+) vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group). Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine (P<0.05). Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Th1 cellular immunity.

Efficient Biolistic Transformation and Regeneration Capacity of an EgTCTP Transgene in Protocorm-like Bodies of Phalaenopsis Orchid

An efficient genetic transformation system using the biolistic method and protocorm-like bodies (PLBs) of Phalaenopsis orchidwas established for introducing the EgTCTP gene obtained from oil palm leaves. A pCAMBIA 1302 vector containing the greenfluorescent protein (mgfp5) as reporter gene and the selectable marker hygromycin phosphotransferase (hpt) gene under the cauliflowermosaic virus (CaMV) 35S promoter were used in this experiment. The transformed PLBs were cultured on MS medium containing 20mg/L hygromycin for 2 months. The surviving PLBs with green fluorescence spots were used to calculate a transformation frequency(93.34%). PLBs containing the transformed EgTCTP gene had the highest percentage of regeneration frequency (95.66%) and numbersof regenerated shoots per explant (3.78±1.89 shoots) compared to the control. The time required for initiation of primordial shoots inthe transformed PLBs (55.22±26.56 days) was much shorter than for the control. Evaluation of the regeneration efficiency, determinedthat the status of the EgTCTP transformants was above average: score=4.04±0.88. The EgTCTP gene was detected in the PLBs overa period of at least 6 months with subculturing every 4 weeks. The stability of the transgenes within the PLBs was confirmed by PCRand this indicated that the transgenes had been integrated into the genome of the transformants. This is the first successful report tointroduce EgTCTP gene into PLBs of Phalaenopsis orchid.

Development of nanoparticle-based magnetic resonance colonography

This study was to develop a novel method of nanoparticle-based MR colonography. Two types of solid lipid nanoparticles (SLNs) were synthesized with loading of (a) gadolinium (Gd) diethylenetriaminepenta acetic acid to construct Gd-SLNs as an MR T1 contrast agent and (b) otcadecylamine-fluorescein-isothiocyanate to construct Gd-fluorescein isothiocyanate (FITC)-SLNs for histologic confirmation of MR findings. Through an in vitro experiment, we first evaluated the size distribution and gadolinium diethylenetriaminepenta acetic acid entrapment efficiency of these SLNs. The SLNs displayed a size distribution of 50-300 nm and a gadolinium diethylenetriaminepenta acetic acid entrapment efficiency of 56%. For in vivo validation, 30 mice were divided into five groups, each of which was administered a transrectal enema using: (i) Gd-SLNs (n=6); (ii) Gd-FITC-SLNs (n=6); (iii) blank SLNs (n=6); (iv) gadolinium diethylenetriaminepenta acetic acid (n=6); and (v) water (n=6). T1-weighted fluid-attenuated inversion-recovery MRI was then performed on mice after transrectal infusion of Gd-SLNs or Gd-FITC-SLNs, which demonstrated bright enhancement of the colonic walls, with decrease in T1 relaxation time. When Gd-FITC-SLNs were delivered, green fluorescent spots were visualized in both the extracelluar space and the cytoplasm through colonic walls under confocal microscopy and fluorescence microscopy. This study establishes the "proof-of-principle" of a new imaging technique, called "nanoparticle-based MR colonography," which may provide a useful imaging tool for the diagnosis of colorectal diseases.

Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors

The coat protein of satellite panicum mosaic virus (SPCP) is known to effectively protect its cognate RNA from deleterious events, and here, we tested its stabilizing potential for heterologous virus-based gene vectors in planta. In support of this, a Potato virus X (PVX) vector carrying the SPMV capsid protein (PVX-SPCP) gene was stable for at least three serial systemic passages through Nicotiana benthamiana. To test the effect of SPCP in trans, PVX-SPCP was co-inoculated onto N. benthamiana together with a Tomato bushy stunt virus (TBSV) vector carrying a green fluorescent protein (GFP) gene that normally does not support systemic GFP expression. In contrast, co-inoculation of TBSV-GFP plus PVX-SPCP resulted in GFP accumulation and concomitant green fluorescent spots in upper, non-inoculated leaves in a temperature-responsive manner. These results suggest that the multifaceted SPMV CP has intriguing effects on virus-host interactions that surface in heterologous systems.

Do nitric oxide donors mimic endogenous NO-related response in plants?

Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 microM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that "donors are compounds that spontaneously break down to release NO" and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.

Stem cell therapy in the aging hearts of Fisher 344 rats: Synergistic effects on myogenesis and angiogenesis

Advanced age is a major risk factor for ventricular dysfunction and reduction of cardiac reserve. Finding novel approaches to prevent and attenuate heart dysfunction associated with advanced age is a major therapeutic challenge. The present study was designed to test whether engrafted embryonic stem cells could improve myocardial function in aging hearts. Cultured mouse embryonic stem cells used for cell therapy were transfected with green fluorescent protein. Aging rats in the cell-treated group received intramyocardial injection of embryonic stem cells. Hemodynamic measurement, myocyte counting, and evaluation of blood flow were performed 6 weeks after cell transplantation. Embryonic stem cell therapy partially improved cardiac reserve, as reflected by the in vivo response to isoproterenol (INN: isoprenaline) stimulation in aging hearts 6 weeks after cell implantation. The functional benefits from engrafted embryonic stem cells were associated with increased myocyte numbers and enhanced left ventricular blood perfusion in the aging heart. The characteristic phenotype of engrafted embryonic stem cells was identified in the transplanted heart on the basis of green fluorescent protein-positive spots that were further demonstrated to differentiate into cardiac tissue with positive staining for cardiac alpha-myosin heavy chain. Regenerating cardiomyocytes and increasing regional blood perfusion in the aging heart after embryonic stem cell transplantation synergistically resulted in improvement of cardiac function. Embryonic stem cell transplantation might hold significant clinical potential in attenuating the progressive decrease of cardiac function associated with advanced aging.

Retrovirus-mediated gene transfer and expression of EGFP in chicken.

Here, we successfully demonstrate expression of the EGFP (enhanced green fluorescence protein) gene in chickens using replication-defective MLV (murine leukemia virus)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G glycoprotein). The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). After 12 days incubation, all of the eight living embryos assayed were found to express this vector-encoded EGFP gene, which was under the control of the RSV (Rous Sarcoma Virus) promoter, in diverse organ tissues, including head, beak, neck, wing, hock, tail, toes, heart, amnion, and yolk sac. Surprisingly, despite the presumed cytotoxicity of EGFP, some embryos hatched and survived and these had prominent green fluorescent spots, both in internal organs and externally.

ARRANGEMENT OF β TUBULIN IN BULL SPERMATOZOA DURING NUCLEI DECONDENSATION

Eukaryotic cells have internal scaffolding of microtubules cytoskeleton that gives them their characteristic shapes. We analyzed by immuno-fluorescence the shift and distribution of tubuline during in vitro bull sperm nuclei swelling by the action of heparin-reduced glutathione physiological decondensing agents. Sperm tubulin display a pattern that shows tubulin fluorescence all over the head, leaving the acrosome tip devoid of tubulin. In the second stage we can observe that the acrosomal zone is practically devoid of fluorescence and a net of fluorescent microtubules that seems to be anchored in the basal plate in the postacrosomal region. It is also possible to observe green spots of tubulin fluorescence in the nucleus periphery, that might represent clusters of chromatin hub-like bodies and/or the microtubule organizing center (MTOC). In the third stage, practically all tubulin moves backwards to the basal plate in the neck region of the sperm nuclei remaining in only the green fluorescence spots in the periphery of the swollen sperm nuclei. The results allow us to assume that tubulin mechanism rearrangement is considered to be necessary for the normal fertilization process.

Receptors for recombinant feline interferon-omega in hemocytes of the Japanese pearl oyster Pinctada fucata martensii.

In a previous study we determined that administration of recombinant feline interferon-omega (rFeIFN-omega) could protect Japanese pearl oysters Pinctada fucata martensii from akoya-virus infection. Our results suggested that rFeIFN-omega enhanced the potential of agranulocytes to phagocytize necrotic cells and to produce collagen fibers that repair the lesions caused by the akoya-virus. In the present study, using an indirect immunofluorescence technique with an anti-rFeIFN-omega rabbit serum, we examined whether hemocytes bear receptors that bind rFeIFN-omega. rFeIFN-omega receptors were present on agranulocytes and bound rFeIFN-omega, and appeared as green fluorescent spots in the cytoplasm under a fluorescent microscope. Around 56% of agranulocytes in Japanese pearl oysters bore the rFeIFN-omega receptors.

Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange.

Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.

Studies on the Movement of Cowpea Mosaic Virus Using the Jellyfish Green Fluorescent Protein

The jellyfish green fluorescent protein (GFP) coding sequence was used to replace the coat protein (CP) genes in a full-length cDNA clone of CPMV RNA-2. Transcripts of this construct were replicated in the presence of RNA-1 in cowpea protoplasts, and GFP expression could be readily detected by fluorescent microscopy. It was not possible to infect cowpea plants with these transcripts, but combined with a mutant RNA-2, in which the 48-kDa movement protein (MP) gene has been deleted infection did occur. With this tripartite virus (CPMV-TRI) green fluorescent spots were visible under UV light on the inoculated leaf after 3 days and a few days later on the higher leaves. These results show that the polyproteins encoded by RNA-2 do not possess an essential function in the virus infection cycle and that there is, contrary to what we have found so far for the proteins encoded by RNA-1, no need for a tight regulation of the amounts of MP and CPs produced in a cell. Subsequently, the GFP gene was introduced between the MP and CP genes of RNA-2 utilizing artificial proteolytic processing sites for the viral proteinase. This CPMV-GFP was highly infectious on cowpea plants and the green fluorescent spots that developed on the inoculated leaves were larger and brighter than those produced by CPMV-TRI described above. When cowpea plants were inoculated with CPMV RNA-1 and RNA-2 mutants containing the GFP gene but lacking the CP or MP genes, only single fluorescent epidermal cells were detected between 2 and 6 days postinoculation. This experiment clearly shows that both the capsid proteins and the MP are absolutely required for cell-to-cell movement.

Production of aflatoxins and other fluorescent metabolites by strains of aspergillus flavus isolated from staple foods and their toxicity to rats

Forty strains of Aspergillus flavus isolated from food marketed in Thailand between 1967 and 1969 were re-isolated for toxicological characterization. Approximately 92% produced aflatoxin B 1 or aflatoxins B 1 and B 2. In acute toxicity tests in weanling male Wistar rats, 87·5% of the orally intubated chloroform extracts (CEX) of the isolates caused at least one death in test groups of five rats, compared with 38% of the ip-administered petroleum ether-insoluble (PEI) fractions. Twenty-four unidentified compounds were produced in varying amounts by eighteen strains of A. flavus. These compounds, detected as blue and green fluorescent spots on thin-layer chromatograms, may have modified the toxic effect of the aflatoxins. The histopathological changes caused by the CEX and PEI fractions were similar, the main ones being haemorrhagic necrosis of the liver and kidney, congestion of pulmonary vessels and hypercellularity of the alveolar septa of the lung, hydropic degeneration of the cardiac muscle and lymphocytic depletion of the spleen.

Quantitative Determination of Chlordiazepoxide and Its Metabolites in Serum by Fluorescence TLC-Densitometry

A sensitive fluorescence TLC-densitometric procedure was developed for the specific determination of chlordiazepoxide (I) and its two metabolites, demoxepam (II) and desmethylchlordiazepoxide (III), in serum. After extraction from serum with ether, I, II, and III were separated by TLC and converted with a sulfuric acid spray to pale blue (Rf 0.63), green (Rf 0.54), and blue (Rf 0.45) fluorescence spots, respectively. Quantitation was accomplished by scanning the plate with a densitometer at 390 (I), 430 (II), and 390 (III) nm. The sensitivities were 0.05 (I), 0.01 (II), and 0.01 (III) μg/ml of serum. The procedure was successfully applied to measurement of I-III in human serum after oral administration of 20mg of chlordiazepoxide hydrochloride.