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PubMed2000

Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast.

Formation and maintenance of specialized plasma membrane domains are crucial for many biological processes, such as cell polarization and signaling. During isotropic bud growth, the yeast cell periphery is divided into two domains: the bud surface, an active site of exocytosis and growth, and the relatively quiescent surface of the mother cell. We found that cells lacking septins at the bud neck failed to maintain the exocytosis and morphogenesis factors Spa2, Sec3, Sec5, and Myo2 in the bud during isotropic growth. Furthermore, we found that septins were required for proper regulation of actin patch stability; septin-defective cells permitted to enter isotropic growth lost actin and growth polarity. We propose that septins maintain cell polarity by specifying a boundary between cortical domains.

Actins↗
PubMed1983

Leu 7+ (HNK-1+) cells. I. Selective compartmentalization of Leu 7+ cells with different immunophenotypes in lymphatic tissues and blood.

In the present immunohistochemical studies, Leu 7+ (HNK-1+, human natural killer and killer) cells were found to occupy preferentially germinal centres of follicles in lymph nodes and tonsils. Leu 7+ cells were also present in germinal-like zones of spleen follicles and in mantle zones of hyperplastic thymus follicles and varied in localization in lymph nodes involved in different types of follicular centre cell-derived malignant lymphomas. Most of the Leu 7+ cells in the follicles expressed the Leu 3 (helper/inducer) marker. Double staining studies of tonsil sheep erythrocyte-rosetting and peripheral blood mononuclear cell suspensions showed that two main, mutually complementary, subpopulations of Leu 7+ cells could be distinguished in both cases, namely Leu 7+/Leu 4+ (subdivided into Leu 2+ (suppressor/cytotoxic) and Leu 3+) and Leu 7+/Leu 4-, including mostly cells with OKM 1 (myelomonocytic) characteristics. Thus, in the tonsil cell suspension the cells with Leu 7+ Leu 3+/OKM 1- immunophenotype strongly predominated, whereas among peripheral blood mononuclear cells Leu 7+Leu 2+/OKM 1- and Leu 7+/OKM 1+ immunophenotypes were mostly observed.

Antibodies, Monoclonal↗
PubMed2006

Regulated compartmentalization of programmed cell death-1 discriminates CD4+CD25+ resting regulatory T cells from activated T cells.

More effective discrimination between CD4+CD25+ regulatory T cells (Treg) and activated T cells would significantly improve the current level of purification of Treg and their therapeutic application. We observed that approximately 90% of Treg (positive for the nuclear transcription factor Forkhead winged helix protein-3 and able to inhibit naive T cell proliferation) isolated from the spleens or lymph nodes of normal mice did not express significant levels of the inhibitory receptor programmed cell death-1 (PD-1) on their surface, but retained PD-1 intracellularly. An identical phenotype was also identified for human CD4+CD25(high) T cells isolated from peripheral blood of healthy volunteers. By contrast, activated T cells expressed high levels of surface PD-1 that paralleled up-regulation of CD25 during effector cell expansion. This distinction allowed us to isolate CD4+CD25+PD-1(-) T cells with suppressive activity from mice immunized with mature allogeneic dendritic cells. Although purification was limited to resting Treg because TCR ligation induced up-regulation of surface PD-1, this strategy nevertheless represents a valuable step toward more definitive characterization of Treg and their improved purification for therapeutic assessment.

Amino Acid Sequence↗
PubMed1987

Glucocorticoid-regulated compartmentalization of cell surface-associated glycoproteins in rat hepatoma cells: evidence for an independent response that requires receptor function and de novo RNA synthesis.

The role of glucocorticoid hormones in the compartmentalization of cell surface-associated mouse mammary tumor virus (MMTV) glycoproteins was examined in M1.54, a cloned line of MMTV-infected rat hepatoma tissue culture cells. The expression of cellular [2-3H]mannose-labeled and cell surface 125I-labeled MMTV glycoproteins was examined throughout a time course of exposure to dexamethasone, a synthetic glucocorticoid. Posttranslational localization of cell surface MMTV glycoproteins required 6 h of exposure to hormone and occurred approximately 4 h after their initial production in an intracellular fraction. This regulated localization to the cell surface correlated with glucocorticoid receptor occupancy and was inhibited by exposure to RU 38486, a powerful antagonist of glucocorticoid-mediated responses. Cell surface immunoprecipitation demonstrated that actinomycin D, an inhibitor of de novo RNA synthesis, prevented regulated expression of cell surface viral glycoproteins, suggesting that newly synthesized cellular components mediate this process. The localization of cell surface MMTV glycoproteins appeared normal in a transcriptional variant (CR1) that produces basal levels of MMTV RNA and glycoprotein precursors in the presence of dexamethasone. Thus, regulated compartmentalization of viral glycoproteins is not an obligate consequence of a critical precursor concentration. Taken together, our results suggest that posttranslational trafficking of cell surface-destined MMTV glycoproteins resulted from an independent glucocorticoid hormone response that required receptor function and de novo RNA synthesis.

Animals↗
PubMed1984

Role of DNase in recovery of plasmid DNA from Clostridium perfringens.

Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol. Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C. perfringens 3626B. Two plasmids (9.4 and 30 megadaltons) were recovered from C. perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels. Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C. perfringens suggested the deleterious DNase activity was not extracellular. Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively). Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP. The variable and irreproducible recovery of plasmid DNA from C. perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.

Clostridium perfringens↗
PubMed2000

Assay of DNA photolyase activity in spinach leaves in relation to cell compartmentation-evidence for lack of DNA photolyase in chloroplasts.

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.

Animals↗
PubMed1996

Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22: their incidence, likely origins, and mechanisms for cell lineage compartmentalization.

Analysis of confined placental mosaicism (CPM) for trisomies 2, 3, 7, 8, 9, 16, and 22, in diagnostic chorionic villus sampling procedures, demonstrates apparent incidences of CPM for individual trisomies of between 9 and 91 cases per 100,000 pregnancies, with trisomy 7 being the most common. More detailed analysis of the percentage of aneuploid cells present, and the distribution of abnormality between the cytotrophoblast and extra-embryonic mesoderm cell lineages, shows a highly specific pattern for each chromosome. Theoretical considerations, in conjunction with direct observations, indicate that the overriding influence on the patterns of cell distribution seen in CPM is the distribution of aneuploid cells laid down during blastogenesis. This in turn reflects closely the origin of mosaicism from either correction of a trisomic conception or post-fertilization somatic error. The pattern of aneuploid cells for each trisomy, as seen at the end of the first trimester and later in pregnancy, can therefore be used to predict the relative contribution of meiotic and mitotic errors to CPM, and hence the likely incidences of uniparental disomy from this source, upd(16)mat being the most common (1 in 10,000 continuing pregnancies). In addition, CPM for trisomies 2, 3, and 8 shows strong evidence of a non-random distribution of aneuploid cells between the different extra-embryonic cell lineages. Analysis of comparable data from spontaneous abortion material repeats this non-random pattern for trisomies 2 and 3, and suggests that a similar phenomenon may also be occurring for trisomy 22. A non-random distribution could be attributable to selection for or against, or intolerance of, particular trisomic cells in certain lineages, but is more probably a result of either cell lineage-specific non-disjunction or consistent uneven compartmentalization of aneuploid cells during blastocyst development.

Chorionic Villi Sampling↗
PubMed1992

Metallothionein biodegradation in rat hepatoma cells: a compartmental analysis aided 35S-radiotracer study.

Disappearance rates, synthesis and biodegradation of rat HTC cell metallothioneins (MT) were examined by measurements of [35S]MT, and expressed by their rate constants in experiments carried out under steady-state conditions, with physiological doses of zinc and copper. Overall rates of disappearance of [35S]MT did not obey first-order kinetics, apparently due to the presence of chemically and/or spatially distinguishable MT (sub)pools. Application of compartmental analysis yielded results indicating that: (a) MT in copper-treated cells is stabler than MT in zinc-treated cells, (b) apparent half-lives for MT disappearance are 22-25 h under the conditions as described, (c) addition of 0.1 mM chloroquine reduces the overall rate of MT disappearance by 25%, (d) total MT should be regarded as significantly consisting of at least two MT sub-forms, i.e., Apo-MT and M-MT and/or cytosolic MT and lysosomal MT, each being depleted and replenished at different rates, (e) mean half-lives for total MT, based on actual degradation rates, were 3-7 h, (f) addition of 0.1 mM chloroquine resulted in both increased synthesis and increased degradation of non-lysosomal MT, the latter probably due to free amino acid depletion in the cells, and (g) MT behaviour may be best examined by combination of tracer experiments (35S-labeled amino acids), simultaneous determination of MT levels in all (sub)MT pools, and application of compartmental analysis.

Animals↗
PubMed1991

Experimental hypersensitivity pneumonitis in the rat: histopathology and T-cell subset compartmentalization.

The pathogenesis of hypersensitivity pneumonitis (HP) appears to depend largely on T-cell specificity and interactions with monocytes/macrophages. We studied T-cell subset populations, defined by surface membrane markers with putative functional correlates, present in lung parenchyma and bronchoalveolar spaces of sensitized LEW rats following acute and chronic inhalational challenges with antigen. Our initial hypothesis was that the CD4+ RT6- (TDH) subset, the effector cell of delayed hypersensitivity, would dominate in HP lesions and that CD8+ RT6+ (TS) or CD8+ CD45R+ (TS) subsets, constituting putative suppressor T-cell populations, would dominate in lungs of animals with resolving lesions. We found, however, a heterogeneous population involving all eight of the T-cell subsets that were evaluated. Percentages of the RT6+ phenotype diminished as T cells moved from peripheral blood to lung. Dominant numbers of T cells in acute HP included CD8+ RT6- (TCYT) and CD8+ CD45R- (TCYT) subsets, with putative cytotoxic T-cell activity, in addition to CD4+ RT6- (TDH) cells. We did not demonstrate increases in T suppressor cells as the disease waned.

Acute Disease↗
PubMed1998

5-Lipoxygenase compartmentalization in granulocytic cells is modulated by an internal bipartite nuclear localizing sequence and nuclear factor kappa B complex formation.

A region of basic amino acids spanning residues 639-656 in the human 5-lipoxygenase sequence resembles a consensus bipartite nuclear localizing sequence. A synthetic peptide consisting of the Kaposi fibroblast growth factor signal sequence fused to the 5-lipoxygenase639-656 bipartite nuclear localizing sequence has a prominent inhibitory effect on 5-lipoxygenase catalysis in granulocytic HL-60 cells activated by calcium ionophor A23187. Recombinant 5-lipoxygenase was not affected by the peptide. The peptide also inhibited redistribution of 5-lipoxygenase from the cytosol to the nuclear membrane of HL-60 cells stimulated by A23187. 5-Lipoxygenase protein was detected in nuclear factor kappaB (NF-kappaB) p65 subunit immunoprecipitate fractions prepared from HL-60 cell lysates. The amount of 5-lipoxygenase protein coimmunoprecipitated by NF-kappaB antiserum was increased following A23187 stimulation. In cells treated with agents that block 5-lipoxygenase translocation to the nucleus, 5-lipoxygenase protein appearing in the NF-kappaB immunoprecipitate was diminished. Our results implicate an internal bipartite nuclear localizing sequence as a regulatory domain that modulates 5-lipoxygenase redistribution and catalysis in granulocytic cells. Additionally, our results suggest that molecular determinants which govern 5-lipoxygenase and NF-kappaB redistribution to the nucleus may be coordinately controlled in granulocytic cells.

Amino Acid Sequence↗
PubMed1992

Primary biphasic lymphoplasmacytic lymphoma of the lung. A mucosa-associated lymphoid tissue lymphoma with compartmentalization of plasma cells in the lung and lymph nodes.

A primary mucosa-associated lymphoid tissue lymphoma of the lung in a 27-year-old man was found. The tumor was composed mainly of centrocyte-like cells and plasma cells. These two components were demarcated sharply from each other, resulting in a zoning or biphasic pattern. This characteristic pattern also was present in the involved regional lymph nodes. Monoclonality of tumor cells was shown immunohistochemically and by in situ hybridization techniques. This article also highlights the use of in situ hybridization in detecting light chain mRNA in paraffin sections.

Adult↗
PubMed1984

Evidence that intracellular synthesis of 5-fluorouridine-5'-phosphate from 5-fluorouracil and 5-fluorouridine is compartmentalized.

L1210 cells were exposed to equitoxic concentrations of [14C]5-fluorouracil and [3H]5-fluorouridine for 4 hours. The RNA from these cells was separated into cytosolic and nuclear fractions, and then further fractionated by chromatography on poly-U Sepharose, Sephadex G-200 and DEAE-cellulose. The ratio of tritium to carbon-14 incorporated into various species of RNA differed by as much as 6-fold, indicating that the respective 5-fluorouridine-5'-monophosphates synthesized from the two precursors are localized in separate pools that do not mix rapidly.

Animals↗
PubMed1994

GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways.

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inositol-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma.

Animals↗
PubMedDate not supplied

Sertoli cell junctional complexes in gossypol-treated neonatal and adult guinea pigs.

The effect of gossypol, an experimental male contraceptive agent, on the development and maintenance of the blood-testis barrier was determined by feeding gossypol daily to prepubertal and adult guinea pigs, and then examining their testes by electron microscopy of thin sections and freeze-fracture replicas. In guinea pigs of 10 to 30 and 10 to 40 days of age that were fed gossypol, impermeable continuous junctional zones did not develop between adjacent Sertoli cells. Compartmentalization of germ cells in the seminiferous epithelium, therefore, was nonexistent. These findings were obtained by use of the sterol-binding polyene, filipin, used as a low molecular weight tracer in combination with freeze-fracture. In general, the seminiferous tubules lacked lumina and spermatogenesis did not progress beyond the pachytene spermatocyte stage. In adult guinea pigs fed gossypol daily for five weeks, continuous zonules at the base of the seminiferous epithelium appeared intact and were impermeable to filipin. Discontinuous zonules found higher in the epithelium showed distensions between interrupted junctional strands and were permeated by filipin. In addition, vacuolated spaces between Sertoli cells and clumps of heterochromatin were conspicuous in some of the Sertoli cell nuclei. Spermatogenesis was disturbed in about 10% of the seminiferous tubules examined. These perturbations included exfoliation of round and elongated spermatids with concomitant formation of multi-nucleated giant cells. Spermatozoa from these adult male guinea pigs were immotile. These findings suggest that, in neonatal animals, gossypol appears to prevent the maturation of Sertoli cells and this effect is expressed as the failure of focal Sertoli cell tight junctional strands to assemble into continuous zonules. In adult animals, gossypol appears to have no effect on the maintenance of the blood-testis barrier.

Age Factors↗
PubMed2005

Actin microridges characterized by laser scanning confocal and atomic force microscopy.

We have characterized the cell surface of zebrafish stratified epithelium using a combined approach of light and atomic force microscopy under conditions which simulate wound healing. Microridges rise on average 100 nm above the surface of living epithelial cells, which correlate to bundles of cytochalasin B-insensitive actin filaments. Time-lapse microscopy revealed the bundles to form a highly dynamic network on the cell surface, in which bundles and junctions were severed and annealed on a time scale of minutes. Atomic force microscopy topographs further indicated that actin bundle junctions identified were of two types: overlaps and integrated end to side T- and Y-junctions. The surface bundle network is found only on the topmost cell layer of the explant, and never on individual locomoting cells. Possible functions of these actin bundles include cell compartmentalization of the cell surface, resistance to mechanical stress, and F-actin storage.

Actin Cytoskeleton↗
PubMed2003

Ectopic LT alpha beta directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase.

Lymph node (LN) function depends on T and B cell compartmentalization, antigen presenting cells, and high endothelial venules (HEVs) expressing mucosal addressin cell adhesion molecule (MAdCAM-1) and peripheral node addressin (PNAd), ligands for naive cell entrance into LNs. Luminal PNAd expression requires a HEV-restricted sulfotransferase (HEC-6ST). To investigate LT alpha beta's activities in lymphoid organogenesis, mice simultaneously expressing LT alpha and LT beta under rat insulin promoter II (RIP) control were compared with RIPLT alpha mice in a model of lymphoid neogenesis and with LT beta-/- mice. RIPLT alpha beta pancreata exhibited massive intra-islet mononuclear infiltrates that differed from the more sparse peri-islet cell accumulations in RIPLT alpha pancreata: separation into T and B cell areas was more distinct with prominent FDC networks, expression of lymphoid chemokines (CCL21, CCL19, and CXCL13) was more intense, and L-selectin+ cells were more frequent. In contrast to the predominant abluminal PNAd pattern of HEV in LT beta-/- MLN and RIPLT alpha pancreatic infiltrates, PNAd was expressed at the luminal and abluminal aspects of HEV in wild-type LN and in RIPLT alpha beta pancreata, coincident with HEC-6ST. These data highlight distinct roles of LT alpha and LT alpha beta in lymphoid organogenesis supporting the notion that HEC-6ST-dependent luminal PNAd is under regulation by LT alpha beta.

Animals↗
PubMedDate not supplied

Novel biphasic elastomeric scaffold for small-diameter blood vessel tissue engineering.

Compliance mismatch, thrombosis, and long culture times in vitro remain important challenges to the clinical implementation of a tissue-engineered small-diameter blood vessel (SDBV). To address these issues, we are developing an implantable elastomeric and biodegradable biphasic tubular scaffold. The scaffold design uses connected nonporous and porous phases as a basis to mimic, respectively, the intimal and medial layers of a blood vessel. Biphasic scaffolds were fabricated from poly(diol citrate), a novel class of biodegradable polyester elastomer. Scaffolds were characterized for tensile and compressive properties, burst pressure, compliance, foreign body reaction (via subcutaneous implantation in rats), and cell distribution and differentiation (via histology, scanning electron microscopy, and immunohistochemistry). Tensile tests, burst pressure, and compliance measurements confirm that the incorporation of a nonporous phase to create a "skin" connected to the porous phase of a scaffold can provide bulk mechanical properties that are similar to those of a native vessel. Compression tests confirm that the scaffolds are soft and recover from deformation. Subcutaneously implanted poly(diol citrate) porous scaffolds produce a thin fibrous capsule and allow for tissue ingrowth. In vitro culture of tubular biphasic scaffolds seeded with human aortic smooth muscle cells (HASMCs) and endothelial cells (HAECs) demonstrates the ability of this design to support cell compartmentalization, coculture, and cell differentiation. The newly formed HAEC monolayer stained positive for von Willebrand factor whereas collagen- and calponin-positive HASMCs were present in the porous phase.

Animals↗