Targeting Homologous Recombination in Notch-Driven C. elegans Stem Cell and Human Tumors

Mammalian NOTCH1-4 receptors are all associated with human malignancy, although exact roles remain enigmatic. Here we employ glp-1(ar202), a temperature-sensitive gain-of-function C. elegans NOTCH mutant, to delineate NOTCH-driven tumor responses to radiotherapy. At ≤20°C, glp-1(ar202) is wild-type, whereas at 25°C it forms a germline stem cell⁄progenitor cell tumor reminiscent of human cancer. We identify a NOTCH tumor phenotype in which all tumor cells traffic rapidly to G2⁄M post-irradiation, attempt to repair DNA strand breaks exclusively via homology-driven repair, and when this fails die by mitotic death. Homology-driven repair inactivation is dramatically radiosensitizing. We show that these concepts translate directly to human cancer models.     

Purified Fc epsilon R+ bone marrow and splenic non-B, non-T cells are highly enriched in the capacity to produce IL-4 in response to immobilized IgE, IgG2a, or ionomycin.

Non-B, non-T cells from spleen and bone marrow cells produce IL-4 in response to cross-linkage of high affinity receptors for Fc epsilon R or Fc gamma RII, and to treatment with calcium ionophores. Cells bearing high affinity Fc epsilon R constituted 1 to 2% of non-B, non-T cells of spleen and of total bone marrow cells from naive donors. In mice whose immune systems had been polyclonally activated by injection with anti-IgD antibodies or had been infected with Nippostrongylus brasiliensis larvae, the frequency of Fc epsilon R+ cells in splenic non-B, non-T cells was also 1 to 2% but in bone marrow from anti-IgD-injected mice donors the frequency was approximately 5%. Cell sorting experiments revealed that all of the capacity to produce IL-4 in response to immobilized IgE or IgG2a or to ionomycin was found in the Fc epsilon R+ fraction. Among the Fc epsilon R+ spleen cells from naive donors, the frequency of IL-4-producing cells was 1/20 to 1/40 whereas in mice that had been injected with anti-IgD or infected with N. brasiliensis, the frequency of IL-4 producing cells in the Fc epsilon R+ population was approximately 1/5.     

Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues

Proenkephalin encodes a group of small peptides with opiate-like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone-derived cells, its modulation by bone active hormones, and the effects of enkephalin-derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin-derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin-derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain. © 1994 Wiley-Liss, Inc.     

Effect of altered sink: source ratio on photosynthetic metabolism of source leaves

When seven crop species were grown under identical environmental conditions, decreased sink:source ratio led to a decreased photosynthetic rate within 1 to 3 days in Cucumis sativus L., Gossypium hirsutum L., and Raphanus sativus L., but not in Capsicum annuum L., Solanum melongena L., Phaseolus vulgaris L., or Ricinus communis L. The decrease was not associated with stomatal closure. In cotton and cucumber, sink removal led to an increase in starch and sugar content, in glucose 6-phosphate and fructose 6-phosphate pools, and in the proportion of ¹⁴C detected in sugar phosphates and UDPglucose following ¹⁴CO₂ supply. When mannose was supplied to leaf discs to sequester cytoplasmic inorganic phosphate, promotion of starch synthesis, and inhibition of CO₂ fixation, were observed in control discs, but not in discs from treated plants. Phosphate buffer reduced starch synthesis in the latter, but not the former discs. The findings suggest that sink removal led to a decreased ratio inorganic phosphate:phosphorylated compounds. In beans ¹⁴C in sugar phosphates increased following sink removal, but without sucrose accumulation, suggesting tighter feedback control of sugar level. Starch accumulated to higher levels than in the other plants, but CO₂ fixation rate was constant for several days.     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves

Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO₂ fixation. Maximal CO₂ fixation rate was obtained when the osmotic potential of both cell isolation and CO₂ fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO₂ fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl⁻ and Na⁺ accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels.     

Rhythmic signalling and entrainment inVespa orientalis larvae: Characterization of the underlying interactions

Rhythmic “circa-second” contrations of larvae of the hornetVespa orientalis, believed to serve as hunger signals, were studied. A considerable degree of coordination among individual larvae, both in frequency and phase of these contractions, has been observed. The oscillations of singly isolated larvae are of short duration, non-constant, with increasing intervals in between and there is a substantial variability in the patterns shown by different larvae. In contrast, the association of two or more larvae leads to enhancement of their periodic behaviour and to (partial) entrainment. Communication among larvae may perhaps be mediated by the sound pulses (“scratching” noises) which are generated by these contractions. We have subjected individual and grouped larvae to external sound pulses and were able to demonstrate: (a) enhancement of rhythmic activity; (b) phase resetting; (c) entrainment to an external oscillator within a range of frequencies; (d) the existence of a subharmonic mode of entrainment. We propose a simple phenomenologic model to account for these larvae responses. Our model assumes the existence of an “energy” variable which declines with time but is upgraded, in a phase-dependent way, by external stimuli. Based in part on work performed by V. Barenholz-Paniry in partial fulfillment of the requirements for the M.Sc. degree from the Sackler Faculty of Medicine, Tel Aviv University, 1986.     

Acclimation of CO(2) Assimilation in Cotton Leaves to Water Stress and Salinity

Cotton (Gossypium hirsutum L. cv Acala SJ2) plants were exposed to three levels of osmotic or matric potentials. The first was obtained by salt and the latter by withholding irrigation water. Plants were acclimated to the two stress types by reducing the rate of stress development by a factor of 4 to 7. CO₂ assimilation was then determined on acclimated and nonacclimated plants. The decrease of CO₂ assimilation in salinity-exposed plants was significantly less in acclimated as compared with nonacclimated plants. Such a difference was not found under water stress at ambient CO₂ partial pressure. The slopes of net CO₂ assimilation versus intercellular CO₂ partial pressure, for the initial linear portion of this relationship, were increased in plants acclimated to salinity of −0.3 and −0.6 megapascal but not in nonacclimated plants. In plants acclimated to water stress, this change in slopes was not significant. Leaf osmotic potential was reduced much more in acclimated than in nonacclimated plants, resulting in turgor maintenance even at −0.9 megapascal. In nonacclimated plants, turgor pressure reached zero at approximately −0.5 megapascal. The accumulation of Cl⁻ and Na⁺ in the salinity-acclimated plants fully accounted for the decrease in leaf osmotic potential. The rise in concentration of organic solutes comprised only 5% of the total increase in solutes in salinity-acclimated and 10 to 20% in water-stress-acclimated plants. This acclimation was interpreted in light of the higher protein content per unit leaf area and the enhanced ribulose bisphosphate carboxylase activity. At saturating CO₂ partial pressure, the declined inhibition in CO₂ assimilation of stress-acclimated plants was found for both salinity and water stress.     

Ligand stimulation of transfected and endogenous growth factor receptors enhances cytokine production by mast cells.

IL-3 dependent mast cell lines produce cytokines in response to Fc receptor cross-linkage or to ionomycin. In this study we have observed that cells pre-cultured in IL-3 produce 10-100 times more cytokine after receptor cross-linkage in comparison with IL-4 pre-cultured cells. Although several hematopoietin receptors, including those for IL-3, IL-4 and EPO, do not contain tyrosine kinase domains, their occupancy with ligand causes tyrosine phosphorylation of specific cellular substrates. Therefore, the contribution of tyrosine kinase activation to the ability of an IL-3 dependent mast cell line, CFTL-15, to produce cytokines was analyzed. The CFTL-15 cells were transfected with growth factor receptors containing ligand-inducible tyrosine kinase domains (EGFR and PDGFR, and CSF-IR) or with the EPOR. All of the transfectants were able to proliferate in response to IL-3 or to their respective growth factor and to produce IL-3 in response to IgE receptor cross-linkage. Stimulation of the EGFR and PDGFR transfectants with their respective ligands resulted in the production of IL-3, IL-6, and GM-CSF. Stimulation of the CSF-1R or EPOR transfectants with growth factor alone failed to induce cytokine production. However, in co-stimulation assays each of the growth factors enhanced the amount of cytokine produced in response to Fc epsilon RI cross-linkage. The ability of these stimuli to induce tyrosine phosphorylation in the transfectants was analyzed. Fc epsilon RI cross-linkage in the transfectants routinely induced the tyrosine phosphorylation of 145, 86 and 72 kDa proteins, with occasional phosphorylation of 55, 52, and 40 kDa proteins.(ABSTRACT TRUNCATED AT 250 WORDS)