Minimizing growth regulators in shoot culture of an endangered plant,Hackelia venusta (Boraginaceae)

Summary Hackelia venusta (Boraginaceae) is an endangered perennial herb endemic to the interior northwestern United States. Because of seed scarcity, micropropagation (anex situ conservation strategy) could produce true-to-type plantlets suitable for reintroduction. We hypothesized that clones of predetermined size could be rapidly produced by supplementing multiplication and rooting media with minimal levels of cytokinin and auxin. Microshoots derived from shoot expants were cultured on Murashige and Skoog (1962) media supplemented with 1% (wt/vol) agar and 0.0001 to 10 μM benzyladenine. Inverse regression estimates on 3 genotypes predicted that a target of 2.5 axillary microshoots per explant would require a minimal level of 0.04±0.02 μM benzyladenine. Culture of 25 genotypes with 0.04 μM benzyladenine resulted in an average of 2.3±0.1 axillary microshoots per explant. Elongated microshoots were transferred to media supplemented with 0.1 to 25 μM indoleacetic acid. Clones rooted from 36% to 100% success after 4 wk in 2.0 μM indoleacetic acid. Plantlets transplantedex vitro with three or more roots survived at 84% versus 46% of plantlets with fewer roots. Up to 84% of the plantlets survived in a planting trial. The data suggest that shoot culture ofHackelia venusta, with minimal growth regulators, can produce axillary microshoots for reintroduction.     

Effects of temperature and food level on growth and development of a planktonic water mite

We analysed the relative effects of food availability and temperature on rates of growth and development of a predatory planktonic water mite, Piona exigua. Growth in length of mites fed Daphnia, Ceriodaphnia and Chydorus was analysed by Gompertz or von Bertalanffy curves; these curves were compared by parallel curve analysis. Growth rates of nymphs and adult female mites increased with temperature; the duration of the imagochrysalis stage decreased. Females grown at 10 °C were smaller at final size than females grown at 15 °C, 18 °C or 22 °C. Females reared at food levels of 15 or 30 prey l⁻¹ grew more slowly and were smaller than those provided with 60 or 120 prey l⁻¹. Nymphs grew more slowly when Daphnia were the only prey, than when smaller prey were available. Food level did not affect nymph growth at 10 °C or 15 °C, but growth at 18 °C or 22 °C may have been slowed at the lowest food levels. Synergistic effects of temperature and food level on nymph growth were apparent only from analysis of growth curves and not from stage duration data.     

Macrophage colony-stimulating factor induces thrombospondin I production by cultured human macrophages

The role of colony-stimulating factors (CSFs) in regulating the synthesis of thrombospondin 1 (TSP1) by cultured human macrophages is investigated. Macrophage (M)-CSF is shown rapidly and transiently to induce two predominant species of TSP1 mRNA. One of these species was 3.2 kb in size and appeared to be specific to M-CSF-stimulated macrophages. Adherent M-CSF-treated macrophages are also shown to express abundant surface cell-associated TSP rapidly when examined by indirect immunofluorescence staining. Granulocyte-macrophage (GM)-CSF induced TSP1 mRNA at a later time point, and this was attributable to the effects of endogenous M-CSF induced by the GM-CSF; the GM-CSF-treated cells did not display surface-associated TSP after 3 hr of treatment. Analysis of the TSP1 protein synthesised by the M-CSF-treated macrophages revealed the expected trimeric form of the molecule. In addition, an unidentified 95-kDa protein was found to be covalently associated with immunoreactive TSP1, and this appeared to be specific to the macrophages as it was not found in TSP1 precipitated from other cell types. It is suggested that the induction of TSP1 by M-CSF may play an important role in the major physiological functions of macrophages. © 1995 Wiley-Liss, Inc.     

Neocentromere-mediated Chromosome Movement in Maize

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences. C urrent models suggest that chromosomes move by a combination of forces generated by microtubule disassembly (Inoue and Salmon, 1995; Waters et al., 1996) and the activity of molecular motors (Vernos and Karsenti, 1996; Yen and Schaar, 1996). Microtubule disassembly generates a constant poleward force; while molecular motors can generate force in either poleward or away-from-pole directions, depending on the characteristics of the motor protein. Both plus and minus end–directed microtubule-based motors are localized to kinetochores (Hyman and Mitchison, 1991). Immunolocalization experiments indicate that mammalian kinetochores contain the minus end– directed motor dynein throughout metaphase and anaphase (Pfarr et al., 1990; Steuer et al., 1990). The kinesin-like proteins CENP-E, which has a transient kinetochore localization in animals, and MCAK, which is localized between the kinetochore plates of mammalian chromosomes, are also thought to generate and/or regulate chromosome movement (Yen et al., 1992; Lombillo et al., 1995; Wordeman and Mitchison, 1995).In addition to the molecular motors on kinetochores, several kinesin-like proteins are localized to chromosome arms (Vernos and Karsenti, 1996). Two subfamilies of arm-based motors have been identified in animals: the NOD subfamily (Afshar et al., 1995; Tokai et al., 1996) and the Xklp1/chromokinesin subfamily (Vernos et al., 1995; Wang and Adler, 1995). Both Nod and Xklp1 are required for positioning chromosomes on the metaphase plate, suggesting that they encode plus end–directed motors (Afshar et al., 1995; Vernos et al., 1995). Other evidence suggests that minus end–directed motors interact with chromosome arms. In the plant Haemanthus, a poleward force acts along chromosome arms during metaphase (Khodjakov et al., 1996), and forces propelling chromosome arms poleward have been detected during anaphase in crane fly spermatocytes (Adames and Forer, 1996). Little is known about how poleward arm motility at metaphase–anaphase affects the fidelity or rate of chromosome segregation.The neocentromeres of maize (Rhoades and Vilkomerson, 1942) provide a particularly striking example of poleward chromosome arm motility. In the presence of Abnormal chromosome 10 (Ab10),¹ heterochromatic DNA domains known as knobs are transformed into neocentromeres and mobilized on the spindle (Rhoades and Vilkomerson, 1942; Peacock et al., 1981; Dawe and Cande, 1996). Knobs are primarily composed of a tandem 180-bp repeat (Peacock et al., 1981) which shows homology to a maize B centromere clone (Alfenito and Birchler, 1993). A characteristic feature of neocentromeres is that they arrive at the spindle poles in advance of centromeres; in extreme cases the neocentromere-bearing chromosome arms stretch towards the poles (Rhoades and Vilkomerson, 1942; Rhoades, 1952). A recently identified mutation (smd1) demonstrates that a trans-acting factor(s) encoded on Ab10 is essential for converting the normally quiescent heterochromatic knobs into active neocentromeres (Dawe and Cande, 1996).Here we use neocentromeres as a model for understanding the mechanisms and importance of nonkinetochore chromosome movement. As a part of our analysis, we developed a four-dimensional system for observing chromosome segregation in living meiocytes. Our experiments were designed to determine (a) how poleward arm motility affects the rate and fidelity of chromosome segregation; and (b) whether the mechanism of neocentromere motility is comparable to the mechanism of kinetochore motility.     

Ribosomal DNA and ITS-2 sequence comparisons as a tool for predicting genetic relatedness

The determination of the secondary structure of the internal transcribed spacer (ITS) regions separating nuclear ribosomal RNA genes of Chlorophytes has improved the fidelity of alignment of nuclear ribosomal ITS sequences from related organisms. Application of this information to sequences from green algae and plants suggested that a subset of the ITS-2 positions is relatively conserved. Organisms that can mate are identical at all of these 116 positions, or differ by at most, one nucleotide change. Here we sequenced and compared the ITS-1 and ITS-2 of 40 green flagellates in search of the nearest relative to Chlamydomonas reinhardtii. The analysis clearly revealed one unique candidate, C. incerta. Several ancillary benefits of the analysis included the identification of mislabelled cultures, the resolution of confusion concerning C. smithii, the discovery of misidentified sequences in GenBank derived from a green algal contaminant, and an overview of evolutionary relationships among the Volvocales, which is congruent with that derived from rDNA gene sequence comparisons but improves upon its resolution. The study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequences, are most useful in systematic and other studies. Received: 14 February 1997 / Accepted: 28 March 1997     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

Changes in key regulatory enzymes of hepatic carbohydrate metabolism after glucose loading of starved rats

When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.     

Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases

Pseudomonas putida produces two lipoamide dehydrogenases, LPD-glc and LPD-val. LPD-val is specifically required as the lipoamide dehydrogenase of branched-chain keto acid dehydrogenase and LPD-glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD-glc has an absorption maximum at 455 nm, but LPD-val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD-glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD-val. LPD-val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.     

Phosphorylation of the microtubule-associated protein MAP2 by GTP.

The chick brain microtubule-associated protein MAP2 can be phosphorylated in vitro to the extent of 12 mol/mol with GTP at the same sites as can be labelled by the cyclic AMP-independent protein kinase utilizing [gamma-32P]ATP as the phosphoryl donor. Consequently, the microtubule protein is chemically modified by the conditions usually employed for studies of microtubule assembly, so that the derived kinetic parameters may not relate to steady-state conditions.