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PubMed1988

[The effect of microenvironment and formation of intra-molecular links on the tertiary structure of cytochrome P-450 LM2].

The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation. Whereas the complete inhibition of oxidation of type II substrate turnover needs two intramolecular cross-links, that of type I substrates necessitates at least seven cross-links. The incorporation of modified and native enzymes into the membrane lipid bilayer at temperatures above the phase transition point results in the enzyme activation. However, in case of the preimmobilized enzyme the activation does not reach the maximal values. Both stabilized and liposome-incorporated cytochrome P-450 can fully be reactivated via the cross-link disulfide bond reduction. No such effect is observed at temperatures below the phase transition point.

Catalysis↗
PubMed1988

The cellular microenvironment and signaling through cell membranes.

The structural and functional aspects of communication between cells have been reviewed, with emphasis on the cell membrane in detection and transductive coupling of oscillating electromagnetic fields in the pericellular environment. Imposed fields are powerful and highly specific tools in manipulation of the sequence of events in membrane transductive coupling. They have revealed nonlinear and nonequilibrium aspects of these interactions. In cerebral tissue, extracellular fields orders of magnitude weaker than the membrane potential can modulate cell firing patterns, entrain EEG rhythms, alter neurotransmitter release and modulate behavioral states. These sensitivities have also been widely detected in non-neural tissues. It is therefore proposed that an intrinsic communication system between cells based on these weak electromagnetic influences may be a general biological property. A three-step model of transductive coupling is presented. First, a highly cooperative modification of calcium binding occurs in the plane of the membrane surface following a focal event at a receptor site. This "amplifying" stage releases substantially more energy than in the initial events. Cerebral extracellular conductance changes accompanying physiological responses may arise in perineuronal fluid with a substantial macromolecular content and calcium ions may modulate perineuronal conductivity. In the second stage, coupling occurs along transmembrane helical proteins and may be mediated by solitons. The third stage couples transmembrane signals to the cytoskeleton and to intracellular enzyme systems, including membrane-bound adenylate cyclase and the protein kinase system of intracellular messengers. Activation of these intracellular systems is calcium-dependent.

Animals↗
PubMed1987

Host origin of the human hematopoietic microenvironment following allogeneic bone marrow transplantation.

The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA-identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.

Adolescent↗
PubMed1988

Effects of transplantation and age on immunohemopoietic cell growth in the splenic microenvironment.

Intact spleens from young adult and aged mice were transplanted into young recipients to compare effects of age and effects of spleen transplantation on hemopoietic and immune functions. Hemopoietic functions of histocompatible spleen transplants were assessed by partial cures of genetically anemic WBB6F1-Sl/Sld recipients, and immune functions were measured as numbers of anti-SRBC PFC(sheep red blood cell plaque-forming cells) and responses to the mitogen PHA (phytohemagglutinin). Spleens from WCB6F1 and WBB6F1 donors at least 28 months old partially corrected anemias in 10 of 28 Sl/Sld recipients, whereas spleens from 5- to 10-month-old donors performed significantly better, partially correcting anemias in 22 of 31 Sl/Sld recipients. B6D2F1 spleens were transplanted from either old or young donors in B6D2F1 recipients to test their ability to support immune-responsive cells. These spleen grafts were much smaller than recipient spleens and contained few anti-SRBC PFC. In contrast WCB6F1-+/+ spleens transplanted in Sl/Sld recipients were much larger, weighing more than the intact spleens of the recipients. Nevertheless when these spleens were from young donors, they contained only about 10% as many anti-SRBC PFC and PHA-responsive cells as did recipient spleens, whereas old donor spleens contained even fewer. Use of splenectomized Sl/Sld recipients did not alter these results. Apparently the effect of transplantation was much more important than age in reducing the spleens' abilities to support immune-responsive cells.

Aging↗
PubMed1986

Resorbing bone stimulates tumor cell growth. A role for the host microenvironment in bone metastasis.

Demineralized extracts of bone matrix and conditioned media from cultured fetal rat calvaria have been reported to contain growth stimulatory activity for bone cells. To investigate the potential role of these local bone growth factors in the development of bone metastases, we chose the Walker 256 carcinosarcoma, a rat mammary tumor which causes osteolytic bone metastases and hypercalcemia. 45Ca-labeled, 19-day fetal Sprague-Dawley rat calvaria were cultured for 96 hours in BGJb medium. Walker cells from ascites tumors or cultures were grown in unconditioned media or in conditioned media harvested from the bone cultures, in the presence of 10% fetal calf serum. Media were changed every 2 days, cells were counted daily for 5 days, and 3H-thymidine uptake into acid insoluble residues was measured. The growth of tumor cells was 5-6-fold greater in conditioned media than in unconditioned media and the effect was dose dependent. Cells cultured in conditioned media demonstrated a approximately 3-fold enhancement of 3H-thymidine incorporation. Generation of growth stimulatory activity correlated with the extent of bone resorption, measured by release of 45Ca from the fetal parietal bones (r = 0.85; P less than 0.001). Conditioned media from bones cultured with 10(-7) M prostaglandin E2 (PGE2) contained greater amounts of growth stimulatory activity than untreated conditioned media, but PGE2 itself did not stimulate tumor cell growth. Addition of 3.5 mM PO4 to bone cultures blocked bone resorption and the generation of growth factors. Growth stimulatory activity was stable to heat (56 C for 30 minutes) and trypsin digestion, with an apparent molecular weight of less than 17,000 daltons by high-performance liquid chromatography. Conditioned medium also stimulated the growth of 13762 rat mammary adenocarcinoma cells, MB-MDA-231 human breast carcinoma cells, TE-85 osteosarcoma cells, a murine fibrosarcoma and rat embryonic fibroblasts, with the most potent effects noted for Walker tumor cells, the TE-85 osteosarcoma, and human breast carcinoma lines. These results suggest a mechanism by which bone resorption could promote the development of skeletal metastasis.

Animals↗
PubMed1978

Dissecting the hematopoietic microenvironment. III. Evidence for a positive short range stimulus for cellular proliferation.

Experiments were carried out with the intent of defining the nature of the microenvironmental defect which severely limits erythropoiesis in the spleen of the S1/S1d mouse. Chimeric spleens, half S1/S1d and half congenic +/+, supported erythropoiesis in the +/+ region but not in the S1/S1d region, regardless of which genotype was the irradiated, marrow-injected host animal. Implants of normal marrow stroma within the spleens of irradiated S1/S1d mice also supported normal proportions of erythrocytic and granulocytic hematopoiesis. Implants of normal spleen stroma, particularly the capsular portion, into unirradiated s1/s1d mice stimulated erythropoiesis originating from S1/S1d stem cells within and in the immediate vicinity of the implant. The evidence suggests a short range, stromal erythropoietic stimulatory factor which is lacking in the S1/S1d.

Anemia, Macrocytic↗