Suppression of mycelia formation by NO produced endogenously in Candida tropicalis

In the experiments reported here we found that enzymatic NO synthesis in the yeast Candida tropicalis resembles the one in animal tissues with respect to the substrate arginine as well as its sensitivity to potential competitive inhibitors. Both, NO produced by the yeast's nitric oxide synthase and NO derived from an artificial donor, suppressed the formation of pseudomycelia. These results suggest to make use of NO as a tool in elucidating the mechanism controlling mycelia generation in this yeast. The apparent K(m) towards oxygen of the yeast's nitric oxide synthase (about 50 microM) was found to be high as compared to the apparent K(m) value of the yeast's respiratory chain (about 170 nM). From this observation it may be concluded that under conditions of little oxygen supply the nitric oxide synthase will unsuccessfully compete for oxygen with respiration. Therefore, the formation of mycelia spontaneously occurring in yeast cultures grown in sealed chambers can be attributed to a reduced internal NO level rather than limited respiratory activity.     

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.     

Cross-adaptation and molecular modeling study of receptor mechanisms common to four taste stimuli in humans

Psychophysical cross-adaptation experiments were performed with two carbohydrates, sucrose (SUC) and fructose (FRU), and two sweeteners, acesulfame-K (MOD) and dulcin (DUL). Seven subjects were asked to match concentrations that elicited the same intensity as a sucrose reference (30 g/l). Cross-adaptation levels were calculated as the ratio of isointense concentrations measured for a given stimulus before and under adaptation. On average, cross-adaptation between SUC and FRU is low and apparently reciprocal. By contrast, cross-adaptation between SUC and MOD is clearly non-reciprocal: SUC adapts MOD significantly (24%, P < 0.005), but MOD fails to adapt SUC (2%, P < 0.79). Significant and reciprocal cross-enhancement is observed between DUL and MOD (approximately -20%, P < 0.03), and also between SUC and DUL (approximately -15%, P < 0.08). In parallel, molecular modeling of the four tastants was performed in order to look for the 12 common binding motifs that were isolated on 14 other tastants in a previous study. SUC and FRU each display 10 out of the 12 binding motifs, whereas DUL and MOD only display four and five distinct motifs respectively and do not have any motif in common. Experimental cross-adaptation levels seem to correlate well with the number of motifs that molecules have in common. FRU and SUC share a majority of binding motifs and correlatively show mutual cross-adaptation. Four motifs of MOD are found among the 10 motifs of SUC, which may explain why SUC cross-adapts MOD but not vice versa. By contrast, DUL and MOD do not share any motif and do not cross- adapt. The various molecular mechanisms that may be responsible for cross-adaptation and/or cross-enhancement are discussed in light of our results.      

The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences

We studied the evolution of the tobacco Tnt1 retrotransposon by analyzing Tnt1 partial sequences containing both coding domains and U3 regulatory sequences obtained from a number of Nicotiana species. We detected three different subfamilies of Tnt1 elements, Tnt1A, Tnt1B, and Tnt1C, that differ completely in their U3 regions but share conserved flanking coding and LTR regions. U3 divergence between the three subfamilies is found in the region that contains the regulatory sequences that control the expression of the well-characterized Tnt1-94 element. This suggests that expression of the three Tnt1 subfamilies might be differently regulated. The three Tnt1 subfamilies were present in the Nicotiana genome at the time of species divergence, but have evolved independently since then in the different genomes. Each Tnt1 subfamily seems to have conserved its ability to transpose in a limited and different number of Nicotiana species. Our results illustrate the high variability of Tnt1 regulatory sequences. We propose that this high sequence variability could allow these elements to evolve regulatory mechanisms in order to optimize their coexistence with their host genome.      

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

THE BALANCE BETWEEN CHASMOGAMY AND CLEISTOGAMY IN COLLOMIA GRANDIFLORA (POLEMONIACEAE)

The extent of cleistogamy (CL) and chasmogamy (CH) was investigated in natural populations of Collomia grandiflora Dougl. ex Lindl. The ubiquitous occurrence of CL flowers was documented in 18 populations from throughout the geographic range of the species. The interrelationship of environmental perturbation, vegetative weight, and the balance between CH and CL flowers was investigated by means of a factorial experiment which varied plant density, incident light and soil mix. An ANOVA revealed that total plant weight increased significantly under conditions of low density, low light and low sand content, with density accounting for most of the variance in plant weight. Interactions involving density were not significant. The number of CH and CL flowers and estimated percent outcrossing increased significantly with increased plant weight, low density, low light, and low sand content, although an analysis of covariance showed that plant weight and density accounted for most of the variance in these traits. Among large, multibranched plants, the proportion of CL flowers increased in a basipetal pattern, whereas most of the CH flowers were restricted to the terminal and upper inflorescences. The smallest plants produced only a single terminal inflorescence comprising entirely CL flowers. The proportion of reproductive effort allocated to outcrossing was dependent upon plant size as estimated from total weight, whereas the proportion allocated to ***selfing was relatively independent. Studies of outcrossed vs. inbred progenies of five populations revealed little evidence for immediate inbreeding depression with respect to plant weight and the number of CH or CL flowers, but indicated that significant quantitative genetic differences exist for these traits among populations of diverse ecological or geographical origin.     

Kinetics of thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.