Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

The Nitrogen Use Efficiency of C(3) and C(4) Plants : III. Leaf Nitrogen Effects on the Activity of Carboxylating Enzymes in Chenopodium album (L.) and Amaranthus retroflexus (L.)

The relationships between leaf nitrogen content per unit area (N_a) and (a) the initial slope of the photosynthetic CO₂ response curve, (b) activity and amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and (c) chlorophyll content were studied in the ecologically similar weeds Chenopodium album (C₃) and Amaranthus retroflexus (C₄). In both species, all parameters were linearly dependent upon leaf N_a. The dependence of the initial slope of the CO₂ response of photosynthesis on N_a was four times greater in A. retroflexus than in C. album. At equivalent leaf N_a contents, C. album had 1.5 to 2.6 times more CO₂ saturated Rubisco activity than A. retroflexus. At equal assimilation capacities, C. album had four times the Rubisco activity as A. retroflexus. In A. retroflexus, a one to one ratio between Rubisco activity and photosynthesis was observed, whereas in C. album, the CO₂ saturated Rubisco activity was three to four times the corresponding photosynthetic rate. The ratio of PEPC to Rubisco activity in A. retroflexus ranged from four at low N_a to seven at high N_a. The fraction of organic N invested in carboxylation enzymes increased with increased N_a in both species. The fraction of N invested in Rubisco ranged from 10 to 27% in C. album. In A. retroflexus, the fraction of N_a invested in Rubisco ranged from 5 to 9% and the fraction invested in PEPC ranged from 2 to 5%.     

Guidelines for the Control of Human Immunodeficiency Virus Infection in Adolescents

Although adolescents account for only 0.4% of reported cases of the acquired immunodeficiency syndrome (AIDS) in the United States, they are sexually active and, therefore, at risk of acquiring human immunodeficiency virus (HIV) infection. To address issues of HIV control in adolescents, we developed guidelines that emphasize education and medical care and deemphasize antibody testing. For adolescents known to be infected with HIV, we recommend no restrictions on access to educational or treatment programs except when their health providers recommend such restrictions to protect them from exposure to opportunistic infections. For adolescents of unknown antibody status with a possible previous exposure to HIV, we recommend that as long as the incidence of HIV infection and clinical AIDS remains low, there should be no restrictions on residential placements and no routine antibody testing.     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Secretion of SPARC by endothelial cells transformed by polyoma middle T oncogene inhibits the growth of normal endothelial cells in vitro.

Endothelioma cells expressing the polyoma virus middle T oncogene induced hemangiomas in mice by the recruitment of nonproliferating endothelial cells from host blood vessels (Williams et al. 1989). I now report that SPARC, a Ca(2+)-binding glycoprotein that perturbs cell-matrix interactions and inhibits the endothelial cell cycle, is produced by endothelioma cells and is in part responsible for the alterations in the morphology and growth that occur when nontransformed bovine aortic endothelial cells are cocultured with endothelioma cells. Normal endothelial cells cocultured with two different middle T-positive endothelial cell lines, termed End cells, exhibited changes in shape that were accompanied by the formation of cell clusters. Media conditioned by End cells repressed proliferation of normal endothelial cells, but enhanced that of an established line of murine capillary endothelium. Radiolabeling studies revealed no apparent differences in the profile of proteins secreted by aortic or capillary cells cultured in End cell conditioned media. Characterization of proteins produced by End cells led to the identification of type IV collagen, laminin, entactin, and SPARC as major secreted products. Although SPARC did not affect the morphology of End or capillary cells, it was associated with overt changes in the shape of aortic endothelial cells. Moreover, SPARC and a synthetic peptide from SPARC domain II inhibited the incorporation of [3H]thymidine by aortic cells, but had minimal to no effect on the capillary endothelial cell line. The inhibition of growth exhibited by aortic endothelial cells cultured in End cell conditioned media could be partially reversed by antibodies specific for SPARC and SPARC peptides. These studies indicate a potential role for SPARC in the generation of hemangiomas by End cells in vivo, a process that requires normal (host) endothelial cells to disengage from the extracellular matrix, withdraw from the cell cycle, migrate, and reassociate into the disorganized cellular networks that comprise cavernous and capillary hemangiomas.     

Culture shock. Selective uptake and rapid release of a novel serum protein by endothelial cells in vitro

A novel protein has been purified from fetal calf serum and from serum-free bovine aortic endothelial cell conditioned culture medium. This protein consists of a single polypeptide chain of reduced Mr 70,000 (70K protein) and was separated from bovine serum albumin and other proteins by ion-exchange chromatography and immunoabsorption on Sepharose-coupled anti-70K protein antiserum. The 70K protein was shown to be structurally and immunologically distinct from bovine serum albumin, alpha-fetoprotein, and vitronectin by one- and two-dimensional peptide mapping, amino acid analysis, and enzyme-linked immunosorbent assay and/or immunoblotting. The 70K protein was located in endothelial cell cytoplasmic granules of irregular size and distribution. Metabolic radiolabeling studies showed that the 70K protein was not a biosynthetic product of these cells; its cytoplasmic location was due to a selective uptake from the fetal calf serum in which the cells were initially grown. After subconfluent cultures of endothelial cells were shifted to serum-free medium, nearly 80% of the total 70K protein that was measurable in the medium was released between 0 and 20 min. Moreover, sparse, rapidly proliferating cells released approximately 18-fold more 70K protein within 2 min as compared to dense, nonproliferating cultures. The concentration of 70K protein in fetal calf serum was estimated to be 400-600 micrograms/ml. Proliferating bovine aortic endothelial cells, 24 h after plating at an intermediate density, released approximately 250 pg of 70K protein/cell within the first 20 min after exposure to serum-free conditions. The data provide evidence for a novel protein in serum which is selectively internalized by endothelial cells in vitro and which in turn is released rapidly under conditions such as osmotic imbalance due to serum removal, or during periods of cellular proliferation, conditions which we term "culture shock."     

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.