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PubMed1987

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line.

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.

Animals↗
PubMed1982

Direct measurement of proton transfer as a probing reaction for the microenvironment of the apomyoglobin heme-binding site.

Aromatic alcohols fluoresce at different wavelengths in their neutral (phiOH*) and anionic (phiO-*) excited states. Consequently, time-resolved fluorescence measurements, at the respective wavelengths, can be used for measuring the rates of proton dissociation and recombination of the excited molecule. As the lifetime of the excited state is very short (a few nanoseconds), the measured reaction is that which takes place in a volume corresponding to the diffusion distance of the proton during the lifetime of the excited state. 8-Hydroxypyrene 1,3,6-trisulfonate (pK = 7.7, pK* = 0.5) is bound to apomyoglobin with a stoichiometry of 1:1. In the bound state its neutral form fluorescence increase 20-fold. The binding affinity is pH-dependent. Two protonatable groups, with pK = 6.5, participate in the stabilization of the negatively charged ligand in the binding site. The ligand is bound only to the apoprotein and is displaced from its site by hemin. Thus we suggest that the ligand is bound to the heme binding site of apomyoglobin. Time-resolved fluorescence of the bound ligand yields the rate constants of proton dissociation and recombination as taking place within the heme binding cavity of apomyoglobin. The rate of proton dissociation is slowed to 7% of the rate measured for the free ligand. Such a slow dissociation indicates a strong interaction of the water in the cavity with the walls [Gutman, M., Huppert, D., and Nachliel, E. (1982) Eur. J. Biochem. 121, 637-642]. The water activity in the site is equivalent to alpha (H2O) = 0.67.

Apoproteins↗
PubMed1982

4-Aminobutyrate aminotransferase. Different susceptibility to inhibitors, microenvironment of the cofactor binding site and distance of the catalytic sites.

The analogues 5-phospho-pyridoxal-aminooxyacetate and 4-vinyl-pyridoxal 5-phosphate inhibit reduced 4-aminobutyrate aminotransferase reconstituted with pyridoxal 5-phosphate, but they do not affect the catalytic activity of the holoenzyme. The binding of the 5-phosphopyridoxal adducts to the holoenzyme was monitored by measuring the fluorescence properties of the protein and the ligands. The presence of nonequivalent binding sites in reduced samples of enzyme containing 1 mol and 1.8 mol P-pyridoxyl residues/mol dimer was detected by steady and nanosecond fluorescence spectroscopy. The spectroscopic properties (fluorescence lifetime tau = 1 ns, polarization = 0.36 and quantum yield = 0.01) of the first molecule of 5-phospho-pyridoxyl are different from the properties of the second molecule of 5-phospho-pyridoxyl covalently bound to the enzyme (tau = 2.1 ns, P = 0.16, QT = 0.1). The spectroscopic properties of pyridoxal 5-phosphate and 5-phospho-pyridoxal-aminooxyacetate bound to different sites on the holoenzyme can be used to deduce proximity relationships between the catalytic sites of 4-aminobutyrate aminotransferase. On the basis of resonance energy transfer measurements, it is proposed that a distance of 27 A (2.7 nm) separates the two catalytic binding sites.

4-Aminobutyrate Transaminase↗
PubMed1993

Microenvironment changes in human blood platelet membranes associated with binding of tissue-type plasminogen activator.

Whole washed platelets were labelled with the free radicals [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16-DOXYL-Ste) or [2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3- oxazolidinyloxy] (5-DOXYL-Ste) and incubated with recombinant tissue-type plasminogen activator (rt-PA). Changes in the membrane fluidity caused by rt-PA were detected by alterations in h+1/h0 calculated from the ESR spectra for 16-DOXYL-Ste and 5-DOXYL-Ste incorporated into the lipid bilayer (h+1 and h0 are the heights of the low-field and middle-field lines of the spectra, respectively). Interaction of rt-PA with both resting and stimulated platelets resulted in increased rigidity of the membrane lipid bilayer as indicated by the reduced value of h+1/h0. This phenomenon can be explained either by conformational changes of membrane receptors caused by the attachment of rt-PA and the subsequent rearrangement of the lipid matrix of platelet membranes, or by the direct association of rt-PA with membrane phospholipids and thus partial embedding of protein molecules into the lipid bilayer restraining lipid mobility.

Blood Platelets↗
PubMed1995

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 1. The partition of hematoporphyrin and protoporphyrin in liposomes.

Temperature-induced fluorescence changes were studied for hematoporphyrin and protoporphyrin, incorporated into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho) or dimiristoylphosphoglycerocholine (Myr2GroPCho). In some cases, cholesterol or cardiolipin were added to the vesicles for better mimicking the lipid composition of biological membranes. The experimental conditions were appropriately chosen in order to reproduce different possible configurations of the porphyrin molecule in lipid membranes: namely, at the polar water/headgroups, headgroups/lipid and lipid/lipid interfaces. A peculiar feature observed in some of the above liposomal systems was the appearance of discontinuities in the Arrhenius plots of the fluorescence quantum yields, with relevant changes of the values of activation energies. These discontinuities were due to an increase of the fluorescence signal in a temperature range corresponding to the transition of the different lipids from the gel-to-liquid crystal state. The observed phenomena are consistent with the formation of non-covalent linear dimers or linear higher aggregates of the porphyrin molecules. The intermolecular contacts required for the formation of these species are favoured by at least three situations: disruption of the ordered lipid structure during the gel-to-liquid crystal phase transition; competition of other polar groups (e.g., the -OH group of cholesterol) with the porphyrin carboxylate groups for the polar phospholipid headgroups; and steric constraints due to overcrowding of porphyrin molecules in a restricted space.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Temperature-induced changes in fluorescence properties as a probe of porphyrin microenvironment in lipid membranes. 2. The partition of hematoporphyrin and protoporphyrin in mitochondria.

The temperature dependence of hematoporphyrin and protoporphyrin fluorescence quantum yields (phi F) was studied after delivery to whole mitochondria or isolated inner (IMM) and outer (OMM) mitochondrial membranes, obtained from liver of Wistar rats. These studies are very sensitive to variations of the porphyrin lipid environment. Before incorporation, the porphyrins were dissolved in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) NaCl/Pi (only hematoporphyrin) or dispersed into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho), sometimes enriched with cholesterol or cardiolipin. Whole mitochondria show higher incorporation capacity of hematoporphyrin and protoporphyrin than isolated IMM and OMM, probably because additional, energy-sensitive transport mechanisms for the porphyrin uptake occur in intact organelles. A small decrease in protoporphyrin uptake is observed in OMM in comparison with IMM; in contrast, the decrease in hematoporphyrin uptake by OMM is rather significant. A comparison between the results obtained with IMM, OMM and whole mitochondria show that both porphyrins, when released to the intact organelles, preferentially localize in the IMM, irrespective of the lipid carrier used. NaCl/Pi-dissolved hematoporphyrin probably interacts with some membrane proteins, due to the similarity of the Arrhenius plots with those obtained for liposome-entrapped human serum albumin/hematoporphyrin complexes which were used as models to mimic hematoporphyrin-membrane protein binding sites. Liposomal hematoporphyrin and protoporphyrin bind to lipid domains. Hematoporphyrin accumulates in specific, localized lipid regions, perhaps in the boundary lipids area surrounding some inner-mitochondrial carriers; protoporphyrin accomodates in more rigid, lipid areas. On these bases, the higher photoactivity of hematoporphyrin, previously observed in mitochondria, in comparison with protoporphyrin, can be easily explained. Formation of linear dimers/aggregates, endowed with higher phi F than that of the monomers, are postulated to occur for both porphyrins only in the inner mitochondrial membrane.

Animals↗
PubMed2006

The microenvironment of vulvar skin in women with symptomatic and asymptomatic herpes simplex virus type 2 (HSV-2) infection.

BACKGROUND: It is not known why some individuals infected with herpes simplex virus type 2 (HSV-2), experience frequent recurrences, while most of those infected have a completely silent infection. OBJECTIVE: We wanted to study if local factors in the skin could explain this difference. DESIGn 21 HSV-2 seropositive patients, 10 with history of >8 clinical recurrences a year (symptomatics) and 11 without symptoms of genital herpes (asymptomatics) were included. All had to answer a questionnaire. With standardised methods, the skin temperature, pH, and the skin barrier function, expressed as transepidermal water loss (TEWL) and skin capacitance, were measured on labium majus and perineum. Culture for bacteria was performed from the same regions. RESULTS AND CONCLUSION: No significant differences in terms of pH and skin barrier function were registered between symptomatic and asymptomatic patients. Asymptomatic patients had a tendency (0.06) to a higher colonisation with lactobacilli on labium majus than symptomatic patients.

Adult↗
PubMed2006

Neocortical microenvironment in patients with intractable epilepsy: potassium and chloride concentrations.

PURPOSE: The regulation of extracellular ion concentrations plays an important role in neuronal function and epileptogenesis. Despite the many studies into the mechanisms of epileptogenesis in human experimental models, no data are available regarding the fluctuations of extracellular potassium ([K(+)](o)) and chloride ([Cl(-)](o)) concentrations, which could underlie seizure susceptibility in human chronically epileptic tissues in vivo. METHODS: By using cerebral microdialysis during surgical resection of epileptic foci, the basic [K(+)](o) and [Cl(-)](o) as well as their changes after epicortical electric stimulation were studied in samples of dialysates obtained from 11 patients by ion-selective microelectrodes. RESULTS: The mean basal values of [K(+)](o) and [Cl(-)](o) in all patients were 3.83 +/- 0.08 mM and 122.9 +/- 2.6 mM, respectively. However, significant differences were observed in the basal levels of both [K(+)](o) and [Cl(-)](o) between different patients. Statistically, no correlation was found between basal [K(+)](o) or [Cl(-)](o) and electrocorticogram (ECoG) spike activity, but in one patient, dramatically lowered baseline [Cl(-)](o) was accompanied by enhanced ECoG spike activity. Application of epicortical electrical stimulation increased [K(+)](o) but not [Cl(-)](o) in all cases. According to the velocity as well as spatial distribution of [K(+)](o) reduction to the prestimulation levels, three different types of responses were observed: slow decline, fast decline, and slow and fast declines at adjacent sites. CONCLUSIONS: These data may represent abnormalities in ion homeostasis of the epileptic brain.

Adolescent↗
PubMedDate not supplied

Stem cell generation and choice of fate: role of cytokines and cellular microenvironment.

Hematopoietic stem cells (HSC) have provided a model for the isolation, enrichment and transplantation of stem cells. Gene targeting studies in mice have shown that expression of the thrombopoietin receptor (TpoR) is linked to the accumulation of HSCs capable to generate long-term blood repopulation when injected into irradiated mice. The powerful increase in vivo in HSC numbers by retrovirally transduced HOX4B, a homeotic gene, along with the role of the TpoR, suggested that stem cell fate, renewal, differentiation and number can be controlled. The discovery of the precise region of the mouse embryo where HSCs originate and the isolation of supporting stromal cell lines open the possibility of identifying the precise signals required for HSC choice of fate. The completion of human genome sequencing coupled with advances in gene expression profiling using DNA microarrays will enable the identification of key genes deciding the fate of stem cells. Downstream from HSCs, multipotent hematopoietic progenitor cells appear to co-express a multiplicity of genes characteristic of different blood lineages. Genomic approaches will permit the identification of the select group of genes consolidated by the commitment of these multipotent progenitors towards one or the other of the blood lineages. Studies with neural stem cells pointed to the unexpected plastic nature of these cells. Isolation of stem cells from multiple tissues may suggest that, providing the appropriate environment/ signal, tissues could be regenerated in the laboratory and used for transplantation. A spectacular example of influence of the environment on cell fate was revealed decades ago by using mouse embryonic stem cells (ES). Injected into blastocysts, ES cells contribute to the formation of all adult tissues. Injected into adult mice, ES cells become cancer cells. After multiple passages as ascites, when injected back into the blastocyst environment, ES- derived cancer cells behaved again as ES cells. More recently, the successful cloning of mammals and reprogramming of transferred nuclei by factors in the cytoplasm of oocytes turned back the clock by showing that differentiated nuclei can be "re-booted" to generate again the stem cells for different tissues.

Journal Article↗
PubMed1992

Modification of human long-term bone marrow cultures: establishment of a functional stromal microenvironment devoid of myeloid progenitors.

Differences in the plastic adhesive properties of bone marrow (BM) cells were used to initiate modified stromal layers (MSL) from long-term cultures by removing non-adherent cells shortly (4 to 18 hours) after initial seeding. Following this early modification, adherent cells generated a confluent layer after 21 days of culture. Cellular characteristics of volume and spontaneous fluorescence determined by flow cytometry showed that the MSL included 82% fibroblastic stromal cells, 8% macrophages and 10% myelomonocytic cells. Furthermore, clonogenic assays revealed that the MSL were devoid of hematopoietic progenitor cells. MSL were found to sustain long-term myelopoiesis for at least 7 weeks from exogenously added hematopoietic progenitors isolated from bone marrow (CD34+ cells), thereby demonstrating their functionality. The present experimental model appears of interest for the study of interactions between defined populations of hematopoietic cells and cells of the adherent layer. Of importance, our present modifications of human long-term bone marrow culture are technically simple and do not involve manipulation of the stromal cells.

Antigens, CD↗
PubMed2004

Microenvironment factors do not afford myeloma cell lines protection from simvastatin.

BACKGROUND: The intensive interactions of myeloma cells (multiple myeloma, MM) with microenvironmental components of the bone marrow contribute significantly to their proliferation and survival. It has been shown that these signals confer drug resistance, delineating their circumvention as a primary objective in disease treatment. This study was designed to assess the effect of some major extracellular factors on the previously established anti-neoplastic response of myeloma cells to simvastatin (Sim). STUDY DESIGN: RPMI8226, U266, and ARH77 seeded in culture plates precoated with fibronectin (FN)/agarose/none were treated with Sim, insulin-like growth factor-I (IGF-I), interleukin-6 (IL-6) or combinations for 5 d. Then we assessed cell morphology, viability (WST1), cell cycle (propidium iodide, PI, staining and flow cytometric analysis), total cell count, and cell death (trypan blue exclusion), and DNA fragmentation. RESULTS AND CONCLUSIONS: Reduced viability was demonstrated with Sim in all treated cell lines with and without co-administration of IGF-I or IL-6 (P < 0.05). The extent of inhibition did not vary between Sim only and combinations (NS). FN did not influence cell response to Sim alone or combined with IL-6/IGF-I (NS). We conclude that IL-6, IGF-I, and FN do not afford myeloma cell lines protection from Sim modulation.

Bone Marrow↗
PubMed1989

Novel cellular interactions and networks involving the intestinal immune system and its microenvironment.

The interactions we have described enable the intestine to respond appropriately to antigenic challenge in an effective and coordinated way. This is of vital importance when one considers the dual role of the intestine as a first line of defence against harmful microorganisms and as the route by which the animal obtains nutrition. Under normal circumstances, these interactions select for an appropriate cell phenotype by providing a network of interactions that contribute to intestinal homeostasis. If there is dysfunction of any component, then other cells will be affected. For example, if down-regulation of the mucosal immune response is not effective, damage to the epithelium, nerves and muscle may occur during an inflammatory response. Similarly, if the integrity of the epithelium is disrupted, damage to the elements of the mucosal immune system may occur. This model would suggest that these interactions must be considered if one wishes to adequately explain diseases such as IBD and design innovative therapeutic regimens. Future interdisciplinary research will shed light on the web of interactions occurring in the intestinal environment and provide a novel view of the respective contributions of the immune system and its local environment to cell differentiation, function and regulation.

Animals↗
PubMed1986

Dynamic variations of the brain cell microenvironment in relation to neuronal hyperactivity.

Neuronal hyperactivity has been shown to cause transient changes in ionic concentrations as well as in the volume of the extracellular space (ECS). During enhanced neuronal activity in the sensorimotor cortex of the cat, increases in the extracellular K+ concentration to a ceiling value of 10 mM have been observed concomitant with decreases in the Na+ concentration of similar magnitude. Simultaneously, the Cl- concentration rose steadily during the enhanced neuronal activity, and the extracellular space decreased by about 30%. A mathematical model allowed the interpretation of these concentration and volume changes as results of K+ release by active neurons in exchange for Na+, removal of K+ from the ECS by spatial glial buffering and movements of KCl into glial cells, as well as cell swelling due to metabolically induced transient increases in cellular osmolarity. Similar mechanisms have also been found to operate in gliotic scar tissue, where glial cells appeared to function the same way as in normal cortex, in rat hippocampal slices, and in preparations studied by other investigators.

Animals↗