Home About us Contact
|
Home About us Contact | ||
|
|
||
Intracellular Environment (intracellular + environment)
Selected AbstractsBiofunctionalized pH-Responsive Microgels for Cancer Cell Targeting: Rational Design,ADVANCED MATERIALS, Issue 1 2006M. Das The design of a drug-delivery system based on bioconjugated, pH-responsive microgels is demonstrated. Microgels loaded with the anticancer drug Doxorubicin are introduced into the HeLa tumor cells by means of receptor- mediated endocytosis. Changes in pH within the intracellular environment induce shrinkage of microgels, triggering the drug release into the cells. The microgel described in this work shows enhanced cytotoxicity to HeLa cells (see Figure). [source] OPTIMIZATION OF PERMEABILIZATION PROCESS FOR LACTOSE HYDROLYSIS IN WHEY USING RESPONSE SURFACE METHODOLOGYJOURNAL OF FOOD PROCESS ENGINEERING, Issue 3 2009GURPREET KAUR ABSTRACT To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation to, -galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost-effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey. PRACTICAL APPLICATION , -Galactosidase is one of the most promising enzymes, which has several applications in the food, fermentation and dairy industry. However, the industrial applications of , -galactosidase have been hampered by the costs involved in downstream processing. The present investigation was focused on developing the low-cost technology for lactose hydrolysis based on permeabilization process. Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. Keeping this in view, lactose hydrolysis in whey has been successfully performed using permeabilized Kluyveromyces marxianus cells. Hydrolysis of lactose using , -galactosidase converts whey into a potentially very useful food ingredient, which has immense applications in food industries. Its use has increased significantly in recent years, mainly in the dairy products and in digestive preparations. Lactose hydrolysis causes several potential changes in the manufacture and marketing of dairy products, including increased solubility, sweetness and broader fermentation possibilities. [source] Intracellular Staphylococcus aureus and antibiotic resistance: Implications for treatment of staphylococcal osteomyelitisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2006J. Kent Ellington Abstract Staphylococcus aureus is responsible for 80% of human osteomyelitis. It can invade and persist within osteoblasts. Antibiotic resistant strains of S. aureus make successful treatment of osteomyelitis difficult. Null Hypothesis: antibiotic sensitivities of S. aureus do not change after exposure to the osteoblast intracellular environment. Human and mouse osteoblast cultures were infected and S. aureus cells were allowed to invade. Following times 0, 12, 24, and 48 h (,± the addition of erythromycin, clindamycin, and rifampin at times 0 or 12 h), the osteoblasts were lysed and intracellular bacteria enumerated. Transmission electron microscopy was performed on extracellular and intracellular S. aureus cells. In mouse osteoblasts, administration of bacteriostatic antibiotics at time 0 prevented the increase in intracellular S. aureus. If the antibiotics were delayed 12 h, this did not occur. When rifampin (bactericidal) was introduced at time 0 to human and mouse osteoblasts, there was a significant decrease in number of intracellular S. aureus within osteoblasts compared to control. If rifampin was delayed 12 h, this did not occur. Significant time-dependent S. aureus structural changes were observed after exposure to the osteoblast intracellular environment. These studies demonstrate that once S. aureus is established intracellularly for 12 h, the bacteria are less sensitive to antibiotics capable of eukaryotic cell penetration (statistically significant). These antibiotic sensitivity changes could be due in part to the observed structural changes. This leads to the rejection of our null hypotheses that the antibiotic sensitivities of S. aureus are unaltered by their location. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell-penetrating abilityJOURNAL OF PEPTIDE SCIENCE, Issue 1 2010Yvonne Turner Abstract A 12-mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell-penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell-penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA-peptide conjugates to be not primarily related to the cell-penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA-peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] The Binding Characteristics and Intracellular Localization of Temoporfin (mTHPC) in Myeloid Leukemia Cells: Phototoxicity and Mitochondrial Damage,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000J. Y. Chen ABSTRACT The state of aggregation of the photosensitizer meso -tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells. [source] Relationship between plasma free fatty acid, intramyocellular triglycerides and long-chain acylcarnitines in resting humansTHE JOURNAL OF PHYSIOLOGY, Issue 24 2009Jill A. Kanaley We hypothesized that plasma non-esterified fatty acids (NEFA) are trafficked directly to intramyocellular long-chain acylcarnitines (imLCAC) rather than transiting intramyocellular triglycerides (imTG) on the way to resting muscle fatty acid oxidation. Overnight fasted adults (n= 61) received intravenous infusions of [U- 13C]palmitate (0400,0830 h) and [U- 13C]oleate (0800,1400 h) labelling plasma NEFA, imTG, imLCAC and im-non-esterified FA (imNEFA). Two muscle biopsies (0830 and 1400 h) were performed following 6 h, overlapping, sequential palmitate/oleate tracer infusions. Enrichment of plasma palmitate was ,15 times greater than enrichment of imTG, imNEFA-palmitate and im-palmitoyl-carnitine. Fatty acid enrichment in LCAC was correlated with imTG and imNEFA; there was a significant correlation between imTG concentrations and imLCAC concentrations in women (r= 0.51, P= 0.005), but not men (r= 0.30, P= 0.11). We estimated that ,11% of NEFA were stored in imTG. imTG NEFA storage was correlated only with NEFA concentrations (r= 0.52, P= 0.004) in women and with (r= 0.45, P= 0.02) in men. At rest, plasma NEFA are trafficked largely to imTG before they enter LCAC oxidative pools; thus, imTG are an important, central pool that regulates the delivery of fatty acids to the intracellular environment. Factors relating to plasma NEFA storage into imTG differ in men and women. [source] An alternative method for delivering exogenous material into developing zebrafish embryosBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2007Vikram Kohli Abstract Non-invasive manipulation of multicellular systems is important for medical and biological research. The ability to introduce, remove, or modify molecules in the intracellular environment is pivotal to our understanding of cellular structure and function. Herein, we report on an alternative method for introducing foreign material into developing embryos using the application of femtosecond (fs) laser pulses. When intense fs laser pulses are focused to a sub-micron spot, transient pores are formed, providing a transport pathway for the delivery of exogenous material into embryonic cells. In this study, zebrafish embryos were used as a model system to demonstrate the non-invasiveness of this applied delivery tool. Utilizing optically induced transient pores chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate), Streptavidin-conjugated quantum dots or DNA (Simian-CMV-EGFP). Pore formation was independent of the targeted location, with both blastomere-yolk interface and blastomere pores competent for delivery. Long-term survival of laser manipulated embryos to pec-fin stage was 89% and 100% for dechorionated and chorionated embryos, respectively. To our knowledge, this is the first report of DNA delivery into zebrafish embryos utilizing fs laser pulses. Biotechnol. Bioeng. 2007;98: 1230,1241. © 2007 Wiley Periodicals, Inc. [source] Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontinCANCER, Issue 8 2009Jami Mandelin PhD Abstract BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies. Cancer 2009. © 2009 American Cancer Society. [source] Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophagesCELLULAR MICROBIOLOGY, Issue 7 2009Tara D. Wehrly Summary The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis ssp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages, to characterize its intracellular biology and identify pathogenic determinants based on their intracellular expression profiles. Phagocytosed bacteria rapidly responded to their intracellular environment and subsequently altered their transcriptional profile. Differential gene expression profiles were revealed that correlated with specific intracellular locale of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of transport and metabolic genes characterized the cytosolic replication stage. Expression of the Francisella Pathogenicity Island (FPI) genes, which are required for intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci encoding putative hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated. Among these, deletion of FTT0383, FTT0369c or FTT1676 abolished the ability of Schu S4 to survive or proliferate intracellularly and cause lethality in mice, therefore identifying novel determinants of Francisella virulence from their intracellular expression profile. [source] Human GCIP interacts with CT847, a novel Chlamydia trachomatis type III secretion substrate, and is degraded in a tissue-culture infection modelCELLULAR MICROBIOLOGY, Issue 10 2007Blandine Chellas-Géry Summary The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D- interacting protein (GCIP). Immunoblot analyses of C. trachomatis -infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis. [source] | |||||||||||||