Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.     

Cytological studies of triploids and their progeny from male-sterile (ms1) soybean

Summary Triploids (2n=3X=60) were obtained from genetic male-sterile (ms1 ms1) soybean [Glycine max (L.) Merr.] plants. Meiosis, pollen fertility, and chromosome number of their progeny were studied. Studies of meiosis in fertile and sterile triploids revealed no distinguishable differences in chromosome associations. Male-sterile plants formed coenocytic microspores characteristic of the ms1 mutant. Restitution of some dyad and tetrad nuclei were observed in male-sterile plants. Chromosomes of the triploids tended to occur in trivalents during diakinesis and metaphase I (MI), but multivalents, bivalents, and univalents also were observed. Average types and frequencies of chromosome associations per cell in diakinesis and MI from 542 pollen mother cells were 0.004 IX + 0.06 VI + 0.002 V + 0.005 IV + 16.99 III + 1.79 II + 5.03 I. Some secondary associations, nonhomologous pairing, and aberrant nucleolar distributions occasionally were observed. Such behavior support the hypothesis of duplicated genomes and the polyploid origin of soybean. Pollen fertility in male-fertile triploid plants (Ms1 ms1 ms1) varied from 57% to 82%, with an average of about 71%. Chromosome numbers of progenies obtained from these fertile triploids varied from 2n=40 to 2n=71, and exhibited a near-random distribution, with the majority (about 60%) being between 56 and 65. Progenies of the fertile triploids gave segregation ratios for the ms1 allele, which confirmed the Ms1 ms1 ms1 genotype.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-11672 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA, Project 2471     

Structural, motional, and kinetic features of the Cu(II)-(L-His)2 complex in aqueous solution

A careful analysis by 1H and 13C FT-NMR on the Cu(II) (L-histidine)2 complex was carried out which allows delineation of structure and dynamics in solution. A mixture of complexes was shown such that 24% of the Cu(II) (L-histidine)2 complex contains both histidines bound in the histaminelike way, while the remaining 76% contains one L-His molecule bound in the histaminelike way and the other L-His molecule bound in the glycinelike way. The motional correlation time and relevant features of the exchange process were also delineated.     

Regulation of photosynthetic carbon assimilation at the cellular level: a review

In green leaves and a number of algae, photosynthetically derived carbon is ultimately converted into two carbohydrate end-products, sucrose and starch. Drainage of carbon from the Calvin cycle proceeds via triose phosphate, fructose 6-phosphate and glycollate. Gluconeogenesis in photosynthetic cells is controlled by light, inorganic phosphate and phosphorylated sugars. Light stimulates the production of dihydroxyacetone phosphate, the initial substrate for sucrose and starch synthesis, and inhibits the degradative pathways in the chloroplast. Phosphate inactivates reactions of synthesis and activates reactions of degradation. Among the phosphorylated sugars a special role is allocated to fructose 2,6-bisphosphate, which is present in the cytoplasm at very low concentrations and inhibits sucrose synthesis directly by inactivating pyrophosphatedependent phosphofructokinase. The synthesis of sucrose plays a central role in the partitioning of photosynthetic carbon. The cytoplasmic enzymes, fructose bisphosphate phosphatase and sucrose phosphate synthase are likely key points of regulation. The regulation is carried out by several effector metabolites. Fructose 2,6-bisphosphate is likely to be the main coordinator of the rate of sucrose synthesis, hence of photosynthetic carbon partitioning between sucrose and starch.Paper presented at the FESP meeting (Strasbourg, 1984)     

Photosynthesis,water use and growth of a C4 grass stand at high CO2 concentration

Leaf photosynthesis rate of the C₄ species Paspalum plicatulum Michx was virtually CO₂-saturated at normal atmospheric CO₂ concentration but transpiration decreased as CO₂ was increased above normal concentrations thereby increasing transpiration efficiency. To test whether this leaf response led growth to be CO₂-sensitive when water supply was restricted, plants were grown in sealed pots of soil as miniature swards. Water was supplied either daily to maintain a constant water table, or at three growth restricting levels on a 5-day drying cycle. Plants were either in a cabinet with normal air (340 mol (CO₂) mol^-1 (air)) or with 250 mol mol^-1 enrichment. Harvesting was by several cycles of defoliation.With abundant water supply high CO₂ concentration did not cause increased growth, but it did not cause an increase in growth over a wide range of growth-limiting water supplies either. Only when water supply was less than 30–50% of the amount used by the stand with a water-table was there evidence that dry weight growth was enhanced by high CO₂. In addition, with successive regrowth, the enhancing effect under a regime of minimal water allocations, became attenuated. Examination of leaf gas exchange, growth and water use data showed that in the long term stomatal conductance responses were of little significance in matching plant water use to low water allocation; regulation of leaf area was the mechanism through which consumption matched supply. Since high CO₂ effects operate principally via stomatal conductance in C₄ species, we postulate that for this species higher CO₂ concentrations expected globally in future will not have much effect on long term growth.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

Radioimmunoassay for fibroblast growth factor (FGF): release by the bovine anterior pituitary in vitro

A radioimmunoassay (RIA) was developed to measure fibroblast growth factor (FGF) using antiserum generated against a synthetic replicate of [Tyr¹⁰]FGF(1–10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro [1], are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr¹⁰]FGF(1–10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N-acetyl-[Tyr¹⁰]FGF(1–10).

Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10⁻⁵ M) or KCl (50 mM). Preincubation of pituitary cells with 17β-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2–3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell growth.     

Enkephalin pseudopeptides: resistance to in vitro proteolytic degradation afforded by amide bond replacements extends to remote sites

Five analogs of leucine enkephalin containing the CH2S group as an amide bond replacement were evaluated with respect to resistance toward degradation by human serum in an HPLC-based assay using both ultraviolet and electrochemical detection. Analogs with the modification at the 1-2, 2-3, 3-4, or 4-5 peptide linkages demonstrated half-lives of 118, 85, 134, and 318 min vs. 12 min for the parent peptide. A pseudopeptide analog with additional D-Ala2 protection had a half-life of greater than 1000 min, while the potent [D-Ala2]-leucine enkephalin analog showed approximately a 10-fold increase in stability. The significant increase in stability for a compound with protection only at the C-terminus suggests that serum enzymes may have greater specificity toward backbone changes than previously realized.