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At least 1,369 records · Page 76Linked to original sources
PubMed2003

Combined hyperthermia and chlorophyll-based photodynamic therapy: tumour growth and metabolic microenvironment.

The effects of combined and simultaneously applied localised 43 degrees C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg(-1) i.v.), (iii) localised HT, (iv) Bchl-ser-PDT+HT. The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied.

Angiogenesis Inhibitors↗
PubMed1997

Photochemical purging of autologous bone marrow grafts: assessment of damage to stem cells and the microenvironment in long-term marrow cultures.

Toxicity of merocyanine 540 (MC540) was assessed in long-term (Dexter-type) bone marrow cultures and in a short-term in vitro clonal assay of fibroblast colony-forming cells (CFU-F). Exposure of freshly explanted mouse bone marrow cells to MC540 (15 micrograms/ml) and white light (fluence: 126 kJ/m2) reduced CFU-F by approximately 2 logs but did not abrogate the cells' capacity to establish and maintain long-term bone marrow cultures. Fat cells were rare or absence in adherent layers established with photosensitized bone marrow cells but the cultures' capacity to generate non-adherent cells, granulocyte/macrophage progenitors (CFU-GM), and early erythroid progenitors (BFU-E) was only moderately (28-36%) reduced.

Animals↗
PubMed2005

The dynamic organization of gene-regulatory machinery in nuclear microenvironments.

Nuclear components are functionally linked with the dynamic temporal and spatial compartmentalization, sorting and integration of regulatory information to facilitate its selective use. For example, the subnuclear targeting of transcription factors to punctate sites in the interphase nucleus mechanistically couples chromatin remodelling and the execution of signalling cascades that mediate gene expression with the combinatorial assembly of the regulatory machinery for biological control. In addition, a mitotic cycle of selective partitioning and sequential restoration of the transcriptional machinery provides a basis for the reassembly of regulatory complexes to render progeny cells competent for phenotypic gene expression. When this intranuclear targeting and localization of regulatory proteins is compromised, diseases, such as cancer, can occur. A detailed understanding of this process will provide further options for diagnosis and treatment.

Animals↗
PubMed1997

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment.

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

Animals↗
PubMedDate not supplied

Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment.

The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 microm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

Animals↗
PubMed2004

p53 cannot be induced by hypoxia alone but responds to the hypoxic microenvironment.

Solid tumors frequently contain hypoxic subregions due to insufficient blood supply. In these domains, cells can undergo p53-dependent apoptosis. Therefore, hypoxia has been implicated as a physiological stimulus for p53 accumulation and activation. In such an environment, p53 mutant cells exhibit a selective growth advantage. Hypoxic regulation of p53 has been proposed to be hypoxia inducible factor (HIF) dependent; however, controversy remains over whether and to what extent low oxygen (O(2)) tension by itself enhances p53 protein stability. Here, we examined the p53 response to hypoxia and hypoxia mimetics in several cell lines expressing different HIF-alpha proteins. Most cells exhibited elevated levels of p53 in response to hypoxia mimetics such as deferoxamine mesylate and CoCl(2), regardless of their HIF-alpha protein expression profile. However, over a range of O(2) levels, from 1.5% to less than 0.02%, we failed to observe p53 accumulation or p53 nuclear translocation in any cell lines tested. Only after treatment with a combination of hypoxia and acidosis/nutrient deprivation did some cells exhibit p53 induction. Our results suggest that, although hypoxia induces p53 accumulation in vivo, secondary effects such as acidosis caused by a hypoxic Pasteur effect (instead of low O(2) by itself) are necessary for p53 accumulation. Therefore, the expression of HIF-1alpha and p53 proteins is not coupled during the cellular hypoxia response.

Apoptosis↗
PubMed2000

Modulation of redox potential in electron transfer proteins: effects of complex formation on the active site microenvironment of cytochrome b5.

The reduction potential of cytochrome b5 is modulated via the formation of a complex with polylysine at the electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the heme propionate contributes to the modulation of the redox potential of cytochrome b5 has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b5. This mutant has been engineered with the aim of restricting water accessibility to the exposed heme edge of cytochrome b5. The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the heme cavity and protects a large section of the heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of cytochrome b5, and with a derivative in which the heme propionates have been converted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the reduction potential of cytochrome b5. In fact, these studies showed that exclusion of water from the complex interface exerts an effect (approximately 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the solvent exposed heme propionate (approximately 30 mV shift).

Binding Sites↗
PubMed2002

Photodimerisation of enones in a clay microenvironment.

The photodimerisation of 2-cyclohexenone, 2-cyclopentenone and their 3-methyl derivatives is studied in the confined environment of clay interlayers. In the photolysis of 2-cyclohexenone, a remarkable regioselectivity is observed in favour of the head-to-head dimer when carried out in cation-exchanged bentonite clays while the dimerisation of 2-cyclopentenone favours the formation of the head-to-tail dimer. The results are explained on the basis of the ground state pre-organisation of enones and also the complexing ability of charge-balancing cations with the carbonyl oxygen of enone. In addition to the observed regioselectivity, other advantages of employing clays in photochemical studies are highlighted.

Journal Article↗
PubMed2003

Photodimerisation of acenaphthylene in a clay microenvironment.

The photochemical dimerisation of acenaphthylene in the presence of various cation-exchanged bentonite clays has been studied. While the cis-dimer is the predominant product when smaller cations are present in the clay interlayer, the presence of heavier atoms in the clay favors the trans-dimer. Synthetic anionic hydrotalcite clays are also found to be efficient for the efficient dimerisation of acenaphthylene.

Journal Article↗
PubMed1987

Differences in the chemical and catalytic characteristics of two crystallographically 'identical' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗
PubMed2004

Lipid rafts: microenvironments for integrin-growth factor interactions in neural development.

The development of a complex multicellular organ such as the nervous system requires precise regulation of cell migration, proliferation and survival. This regulation in turn requires the integration of long-range signals, such as growth factors, with short-range cues that define the precise location and cellular neighbours for any given cell. This short review examines one integrative mechanism, integrin-growth factor receptor interactions, and explores the role of lipid rafts in the molecular mechanisms that underlie the receptor interactions.

Animals↗
PubMed2002

Laminin gamma2 chain as a stromal cell marker of the human bone marrow microenvironment.

Laminins are large heterotrimeric molecules consisting of alpha, beta and gamma chains. At present, five alpha chains, three beta chains and three gamma chains have been characterized. Laminin-5 (alpha3beta3gamma2) is the only isoform known to date which contains a gamma2 chain. In human bone marrow, non-haematopoietic stromal cells expressed the laminin gamma2 chain, whereas bone marrow mononuclear cells did not. Co-localization of the gamma2 chain was detected with the laminin alpha4 and alpha5 chains, and co-immunoprecipitation studies revealed a new isoform consisting of alpha5, beta2 and gamma2 chains. The laminin gamma2 chain was also co-localized with alpha-sm-actin in bone marrow, but it was not expressed in endothelial cells or megakaryocytes, indicating that the gamma2 chain is exclusively expressed in vivo in bone marrow stromal cells. The laminin gamma2 chain containing isoform LN-5 was shown to be an adhesive substrate for a small subpopulation of bone marrow mononuclear cells and also for peripheral blood platelets. Taken together, these results indicate that (I) laminin isoforms containing the gamma2 chain can act as adhesive substrates for human haematopoietic cells, and (II) the laminin gamma2 chain can be used as a specific marker molecule for human bone-marrow-derived stromal cells.

Biomarkers↗
PubMed1996

Lysis of pulmonary fibroblasts by lymphokine (IL-2)-activated killer cells--a mechanism affecting the human lung microenvironment?

In this study we investigated whether IL-2-activated killer cells may bind and exert lytic activity against non-transformed lung fibroblasts. We demonstrated that human lymphokine-activated killer (LAK) cells generated in vitro following incubation with recombinant IL-2 of either peripheral blood mononuclear cells (PB-LAK) or lymphocytes obtained from bronchoalveolar lavage (BAL-LAK), but not resting cells, can lyse normal lung fibroblasts obtained from transbronchial lung biopsies in a 4-h 51Cr release assay. Both autologous and allogeneic fibroblasts were consistently lysed by LAK cells, thus suggesting that the phenomenon we observed is not MHC-restricted. Since fibroblasts can bind IL-2 through specific receptors, we evaluated whether long-term culture with rIL-2 could modulate the susceptibility to lysis of target cells. Our data showed that autologous fibroblasts were more resistant to lysis than allogeneic fibroblasts when they were cultured with rIL-2. Since LAK cells have been demonstrated to release a series of different immunomodulatory cytokines, we evaluated the effect of short-term incubation of fibroblasts with different factors, including IL-1, IL-2, IL-3, IL-4, IL-6, tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma), on the binding and the lysis mediated by LAK cells. These cytokines were not directly cytotoxic on fibroblasts. Only IFN-gamma was found to have a significant protective effect against the lysis. Our data support the concept that a self-directed cytotoxicity against pulmonary fibroblasts is generated during lymphocyte activation with rIL-2.

Cells, Cultured↗
PubMed1998

Changes to the cytokine microenvironment in the genital tract mucosa of HIV+ women.

As previous studies have indicated that genital tract mucosal T cell function may be impaired in HIV infection, we investigated the T cell cytokine mRNA in the genital tract mucosa of HIV-infected women to determine if there are alterations in the cytokine profile which may explain the T cell impairment. The in situ hybridization technique was used to investigate the T helper-1 (Th1: IL-2, interferon-gamma (IFN-gamma)) and Th2 cytokine (IL-4, IL-5, IL-10) mRNA profile in cervical biopsies from 10 HIV+ and 10 HIV- subjects. Cervical intraepithelial neoplasia (CIN) and genital infection had previously been excluded and the distribution of immunocompetent cells within the cervical mucosa was known for each subject. Non-parametric tests were used to compare the optical density (OD) of cytokine mRNA in the HIV+ and HIV- groups. Comparisons were also made between peripheral CD4 lymphocyte counts, cervical CD4/CD8 T lymphocyte ratios and cytokine mRNA OD in HIV+ subjects. The HIV+ women had significantly higher mRNA OD for the Th2 cytokines IL-4, IL-5 and IL-10 than HIV women. There was also significantly lower IL-2 mRNA OD in the former group. HIV+ women had lower IFN-gamma mRNA than HIV- women, but the difference was not statistically significant. There was no correlation between cytokine mRNA OD and peripheral CD4 count or cervical CD4/CD8 ratio. The predominance of Th2 cytokines, which are immuno-inhibitory, in the cervical mucosa of HIV+ women may underlie the impaired cytotoxic potential observed in the CD8+ T lymphocytes and may contribute to the susceptibility of HIV-infected women to recurrent genital tract infections and cervical neoplasia.

Adult↗
PubMed1998

Differential distribution of Fc gamma RIIIa in normal human tissues and co-localization with DAF and fibrillin-1: implications for immunological microenvironments.

Fc gamma RIIIa is a cytokine-inducible IgG Fc receptor implicated in the activation of macrophages by immune complexes. Differential expression of Fc gamma RIIIa by macrophages in different tissues may therefore modulate local immune responsiveness. Fc gamma RIIIa expression in normal human tissues was assessed semiquantitatively using microdensitometry. Synovial intimal, serosal, alveolar, salivary gland and placental macrophages, Kupffer cells, and macrophages in mechanically stressed dermis expressed high levels of Fc gamma RIIIa. Less consistent expression was seen in skeletal muscle and lymphoid organs. No significant expression was observed in brain, thyroid, spine, intestine, myocardium, prostate, uterus, flexor forearm dermis, uterus, or kidney. Staining for Fc gamma RIII was also observed on extracellular matrix, and co-localized with both complement decay-accelerating factor and fibrillin-1. It is proposed that differential levels of both cellular and extracellular Fc gamma RIIIa, by modulating the response to immune complexes, may contribute to relative tissue susceptibility to infection and autoimmune disease.

CD55 Antigens↗