Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a β-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution. Received: 18 April 2000 / Accepted: 10 July 2000     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

Comparative effects of ethylene and cyanide on respiration, polysome prevalence, and gene expression in carrot roots

Treatment of carrot roots (Daucus carota L.) with 10 microliters per liter ethylene in O₂ evokes a three- to four-fold increase in polysome prevalence and associated poly(A)⁺ RNA. The increase in polysome prevalence is attended by a similar change in CO₂ evolution. The increase in polysomal poly(A)⁺ mRNA constitutes primarily a generic increase in constitutive mRNAs as assayed by in vitro translation. However, changes in the relative abundance of several in vitro translatable ethylene specific mRNAs do occur.

Cyanide, at concentrations which inhibit cytochrome oxidase, initiates a respiratory rise very similar in kinetics and magnitude to that evoked by ethylene. Moreover, the combined treatment with cyanide and ethylene evokes a respiratory response resembling that caused by ethylene or cyanide alone. Nevertheless, cyanide, in the presence of ethylene, significantly inhibits the increase in polysome prevalence and new gene expression associated with ethylene treatment of carrot roots. Separation of in vitro translation products by one-dimensional and two-dimensional gel electrophoresis shows that several new in vitro translation products appear in cyanide-treated carrots different from those evoked by ethylene. Engagement of the less energy efficient alternative electron transport path by cyanide may be responsible for inhibition of the normal ethylene associated increase in polysome prevalence and new gene expression. The implications of these results on regulation of respiratory metabolism are discussed and compared with the results for similar experiments with avocado fruit (Tucker and Laties 1984 Plant Physiol 74: 307-315) in which cyanide does not inhibit an ethylene educed increase in polysome prevalence and change in gene expression.

     

Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense Rab11 GTPase gene

A cDNA clone from tomato fruit encodes a protein with strong homology with the rab11/YPT3 class of small GTPases that is thought to be involved in the control of protein trafficking within cells. The gene, LeRab11a, showed a pattern consistent with a single copy in DNA gel blots. The corresponding mRNA was developmentally regulated during fruit ripening, and its expression was inhibited in several ripening mutants. Its reduced expression in the Never-ripe mutant indicates that it may be induced by ethylene in fruit. The ripening-induced expression in tissues that are undergoing cell wall loosening immediately suggests a possible role in trafficking of cell wall-modifying enzymes. The message also was produced in leaves and flowers but not in roots. Antisense transformation was used to generate a "mutant phenotype." Antisense fruit changed color as expected but failed to soften normally. This was accompanied by reduced levels of two cell wall hydrolases, pectinesterase and polygalacturonase. There were other phenotypic effects in the plants, including determinate growth, reduced apical dominance, branched inflorescences, abnormal floral structure, and ectopic shoots on the leaves. In some plants, ethylene production was reduced. These data suggest an alternative or additional role in exocytosis or endocytosis of homeotic proteins, hormone carriers, or receptors.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Responsiveness of the olfactory receptor cells in dog to some odors

A preparation has been developed in the dog which allows recording the electrical activity from an olfactory nerve twig containing the axons of a small group of olfactory receptor cells. The dog's response to n-pentyl acetate is vigorous and stable, like that of other air-breathing animals. The dog's response magnitude dependence on the nasal flow rate was noticeable for n-pentyl acetates, but not so great as for n-butyric acid. The response to n-butyric acid strongly depends on the nasal flow. The start of the nasal air flow caused an increase of neural activity, which is called flow response. The results show that the nasal flow rate is a very important factor which determines the response to odors. Methyl p-hydroxybenzoate is known as a dog's pheromone, however, this odor caused the feeble response in the electrical activity of the dog's olfactory receptor cells. The differences may be dependent on several factors.     

Linker region of Akt1/protein kinase Balpha mediates platelet-derived growth factor-induced translocation and cell migration

The phosphatidylinositol 3-kinase (PI3K) signaling pathway(s) is activated by a variety of agonists to regulate cell migration. Here, we show that the stimulation of mouse embryonic fibroblasts with platelet-derived growth factor (PDGF) induces migration in a PI3K-dependent manner. Cells lacking Akt1/PKBalpha exhibit impaired migration and peripheral ruffling in response to PDGF stimulation, whereas cells lacking Akt2/PKBbeta are normal. In addition, over-expression of Akt1/PKBalpha but not Akt2/PKBbeta is sufficient to restore PDGF-induced cell migration in an Akt1/PKBalpha and Akt2/PKBbeta deficient background. In response to PDGF stimulation, Akt1/PKBalpha selectively translocates to membrane ruffles, however, this localization is abrogated by substituting the linker region of Akt2/PKBbeta. Similarly, expression of an Akt2/PKBalpha chimera containing the linker region of Akt1/PKBalpha restored PDGF-induced migration in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta. Finally, over-expression of constitutively active Rac rescues PDGF-induced migration defects in cells lacking Akt1/PKBalpha. Given these results, we suggest that Akt1/PKBalpha controls cell migration by selectively translocating to the leading edge and activating Rac.     

Genetic Variants in Epidermal Growth Factor Receptor Pathway Genes and Risk of Esophageal Squamous Cell Carcinoma and Gastric Cancer in a Chinese Population

The epidermal growth factor receptor (EGFR) signaling pathway regulates cell proliferation, differentiation, and survival, and is frequently dysregulated in esophageal and gastric cancers. Few studies have comprehensively examined the association between germline genetic variants in the EGFR pathway and risk of esophageal and gastric cancers. Based on a genome-wide association study in a Han Chinese population, we examined 3443 SNPs in 127 genes in the EGFR pathway for 1942 esophageal squamous cell carcinomas (ESCCs), 1758 gastric cancers (GCs), and 2111 controls. SNP-level analyses were conducted using logistic regression models. We applied the resampling-based adaptive rank truncated product approach to determine the gene- and pathway-level associations. The EGFR pathway was significantly associated with GC risk (P = 2.16×10⁻³). Gene-level analyses found 10 genes to be associated with GC, including FYN, MAPK8, MAP2K4, GNAI3, MAP2K1, TLN1, PRLR, PLCG2, RPS6KB2, and PIK3R3 (P<0.05). For ESCC, we did not observe a significant pathway-level association (P = 0.72), but gene-level analyses suggested associations between GNAI3, CHRNE, PAK4, WASL, and ITCH, and ESCC (P<0.05). Our data suggest an association between specific genes in the EGFR signaling pathway and risk of GC and ESCC. Further studies are warranted to validate these associations and to investigate underlying mechanisms.     

Effect of pain location on spatial reorganisation of muscle activity

IntroductionWe aimed to determine whether the changes in muscle activity (in terms of both gross electromyography (EMG) and motor unit (MU) discharge characteristics) observed during pain are spatially organized with respect to pain location within a muscle which is the main contributor of the task.MethodsSurface and fine-wire EMG was recorded during matched low-force isometric plantarflexion from soleus (from four quadrants with fine-wire EMG and from the medial/lateral sides with surface EMG), both gastrocnemii heads, peroneus longus, and tibialis anterior. Four conditions were tested: two control conditions that each preceded contractions with pain induced in either the lateral (Pain_L) or medial (Pain_M) side of soleus.ResultsNeither the presence (p = 0.28) nor location (p = 0.19) of pain significantly altered gross muscle activity of any location (lateral/medial side of soleus, gastrocnemii, peroneus longus and tibialis anterior). Group data from 196 MUs show redistribution of MU activity throughout the four quadrants of soleus, irrespective of pain location. The significant decrease of MU discharge rate during pain (p < 0.0001; Pain_L: 7.3 ± 0.9–6.9 ± 1.1 Hz, Pain_M: 7.0 ± 1.1 to 6.6 ± 1.1 Hz) was similar for all quadrants of the soleus (p = 0.43), regardless of the pain location (p = 0.98). There was large inter-participant variation in respect to the characteristics of the altered MU discharge with pain.ConclusionResults from both surface and fine-wire EMG recordings do not support the hypothesis that muscle activity is reorganized in a simple systematic manner with respect to pain location.