934 resultados para Plant-tissue culture
Resumo:
To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.
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Enteropathogenic Escherichia coli (EPEC) causes a characteristic histopathology in intestinal epithelial cells called the attaching and effacing lesion. Although the histopathological lesion is well described the bacterial factors responsible for it are poorly characterized. We have identified four EPEC chromosomal genes whose predicted protein sequences are similar to components of a recently described secretory pathway (type III) responsible for exporting proteins lacking a typical signal sequence. We have designated the genes sepA, sepB, sepC, and sepD (sep, for secretion of E. coli proteins). The predicted Sep polypeptides are similar to the Lcr (low calcium response) and Ysc (yersinia secretion) proteins of Yersinia species and the Mxi (membrane expression of invasion plasmid antigens) and Spa (surface presentation of antigens) regions of Shigella flexneri. Culture supernatants of EPEC strain E2348/69 contain several polypeptides ranging in size from 110 kDa to 19 kDa. Proteins of comparable size were recognized by human convalescent serum from a volunteer experimentally infected with strain E2348/69. A sepB mutant of EPEC secreted only the 110-kDa polypeptide and was defective in the formation of attaching and effacing lesions and protein-tyrosine phosphorylation in tissue culture cells. These phenotypes were restored upon complementation with a plasmid carrying an intact sepB gene. These data suggest that the EPEC Sep proteins are components of a type III secretory apparatus necessary for the export of virulence determinants.
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We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.
Resumo:
Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program.
Resumo:
Acid extracts and a resultant fraction from solid-phase extraction (SPE) of Romalea guttata crop and midgut tissues induce sorghum (Sorghum bicolor var. Rio) coleoptile growth in 24-h incubations an average of 49% above untreated controls. When combined with plant auxin, indole-3-acetic acid (IAA), the SPE fraction shows a synergistic reaction, yielding increases in coleoptile growth that average 295% above untreated controls and 8% above IAA standards. The interaction lowered the point of maximum sensitivity of IAA 3 orders of magnitude, resulting in a new IAA physiological set point at 10(-7) g/ml. This synergism suggests that contents in animal regurgitants making their way into plant tissue during feeding may produce a positive feedback in plant growth and development following herbivory. Such a process, also known as reward feedback, may exert major controls on ecosystem-level relationships in nature.
Resumo:
Jasmonic acid (JA) is a naturally occurring growth regulator found in higher plants. Several physiological roles have been described for this compound (or a related compound, methyl jasmonate) during plant development and in response to biotic and abiotic stress. To accurately determine JA levels in plant tissue, we have synthesized JA containing 13C for use as an internal standard with an isotopic composition of [225]:[224] 0.98:0.02 compared with [225]:[224] 0.15:0.85 for natural material. GC analysis (flame ionization detection and MS) indicate that the internal standard is composed of 92% 2-(+/-)-[13C]JA and 8% 2-(+/-)-7-iso-[13C]JA. In soybean plants, JA levels were highest in young leaves, flowers, and fruit (highest in the pericarp). In soybean seeds and seedlings, JA levels were highest in the youngest organs including the hypocotyl hook, plumule, and 12-h axis. In soybean leaves that had been dehydrated to cause a 15% decrease in fresh weight, JA levels increased approximately 5-fold within 2 h and declined to approximately control levels by 4 h. In contrast, a lag time of 1-2 h occurred before abscisic acid accumulation reached a maximum. These results will be discussed in the context of multiple pathways for JA biosynthesis and the role of JA in plant development and responses to environmental signals.
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Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy. In the adult human brain, six isoforms of tau are expressed that differ by presence or absence of the second of the four semiconserved repeats. As a consequence, half of the tau isoforms have three repeats (3R tau), whereas the other half has four repeats (4R tau). Site-directed spin labeling of recombinant tau in conjunction with electron paramagnetic resonance spectroscopy was used to obtain structural insights into tau filaments. The studies showed that the filaments of 4R tau and 3R tau share a highly ordered core structure in the third repeat with parallel, in-register arrangement of beta-strands. This structure in 3R and 4R is conserved regardless of whether full-length isoforms (htau40 and htau23) or truncated constructs (K18 and K19) are used. When mixed, 3R tau and 4R tau coassembled into heterogeneous filaments. Hence, these findings indicate that there are at least three compositionally distinct types of filaments: homogeneous 3R tau, homogeneous 4R tau, and heterogeneous 3R/4R tau. In vitro experiments show that the seeded filament growth, a prerequisite for tau spreading in tissue culture and brain, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms whereas seeds of 4R tau can recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. Conformational studies at the molecular level of tau filaments were done using Double electron-electron resonance spectroscopy, which allows the determination of distances between pairs of spin labels. These studies revealed structural differences between filaments of 3R tau and 4R tau. Furthermore, they indicated that 4R tau assumed the conformation of 3R tau when templated on 3R tau seeds. Our measurements have also provided insights into the heterogeneity of tau filament structure. Conformational differences due to variation in filament composition and seeding properties of tau filaments have shown that they are structurally polymorphic in nature. This structural polymorphism of tau filaments has widespread implications in understanding and treatment of neurodegenerative diseases.
Resumo:
A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P < 0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS). Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding. To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed. Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells. The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal toot for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.
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The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.
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The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.
Resumo:
Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly( beta- hydroxybutyrate- co- beta- hydroxyvalerate) ( PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self- renewal capacity, and osteogenic potential of osteoblast- like cells ( MC3T3- E1 S14) when cultured on PHBV films compared with tissue culture polystyrene ( TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase ( ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle- shaped MC3T3- E1 S14 cells made cell - cell and cell - substrate contact. Time- dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro- collagen alpha 1( I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa- 1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.
Resumo:
The relatively low numbers and sporadic pattern of incidence of the acetic acid bacterium Gluconacetobacter sacchari with the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae) over time and from different sugarcane-growing regions do not indicate that Glac. sacchari is a significant commensal of the PSMB, as has been previously proposed. This study was conducted to investigate the hypothesis that Glac. sacchari is, like its closest relative Glac. diazotrophicus, an endophyte of sugarcane (Saccharum officinarium L.). In this study, both Glac. sacchari and Glac. diazotrophicus were isolated from internal sugarcane tissue, although the detection of both species was sporadic in all sugarcane-growing regions of Queensland tested. To confirm the ability of Glac. sacchari to live endophytically, an experiment was conducted in which the roots of micropropagated sugarcane plantlets were inoculated with Glac. sacchari, and the plantlets were subsequently examined for the presence of the bacterium in the stem cells. Pure cultures of Glac. sacchari were grown from homogenized surface sterilized sugarcane stems inoculated with Glac. sacchari. Electron microscopy was used to provide further conclusive evidence that Glac. sacchari lives as an endophyte in sugarcane. Scanning electron microscopy of (SEM) sugarcane plantlet stems revealed rod-shaped cells of Glac. sacchari within a transverse section of the plantlet stem cells. The numbers of bacterial cells inside the plant cell indicated a successful infection and colonization of the plant tissue. Using transmission electron microscopy, (TEM) bacterial cells were more difficult to find, due to their spatial separation. In our study, bacteria were mostly found singularly, or in groups of up to four cells inside intercellular spaces, although bacterial cells were occasionally found inside other cells.
Resumo:
Cut Geraldton waxflower (Chamelaucium uncinatum Schauer) flowers are often infected with Botrytis cinerea. Release of infection from quiescence can cause ethylene production by invaded host tissues and result in flower abscission. Postharvest floral organ abscission is a major problem for the commercial waxflower industry. Methyl jasmonate (MeJA) occurs naturally in plant tissue and has a signalling role in eliciting induced systemic resistance against disease. MeJA treatments have been shown to suppress B. cinerea infecting cut rose flowers. The present experiments investigated the potential of exogenous MeJA treatments for B. cinerea management on harvested waxflower. MeJA treatments of 10 and 100 L liquid MeJA/L of air applied to cv. Purple Pride and 1 L MeJA/L to cv. Mullering Brook gave reductions in disease severity for uninoculated stems. However, concentrations of 100 L MeJA/L applied to Purple Pride in addition to 1 and 10 L MeJA/L applied to Mullering Brook increased the incidence of floral organ fall. Flower abscission upon treatment with MeJA may be due to induced systemic resistance-associated upregulation of ethylene biosynthesis. MeJA treatments had no direct effect on B. cinerea hyphal elongation in vitro. Collectively, these results show that while MeJA treatment may elicit defence in waxflower against Botrytis, the chemical also causes floral organ fall. Thus, exogenous MeJA treatments do not have potential for B. cinerea management on harvested waxflower.