Anther developmental defects in Arabidopsis thaliana male-sterile mutants

 We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis. Received: 15 October 1998 / Revision accepted: 19 November 1998     

Leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats

The branched-chain amino acid leucine stimulates muscle protein synthesis in part by directly activating the mTOR signaling pathway. Furthermore, leucine, if given in conjunction with resistance exercise, enhances the exercise-induced mTOR signaling and protein synthesis. Here we tested whether leucine can activate the mTOR anabolic signaling pathway in uremia and whether it can enhance work overload (WO)-induced signaling through this pathway. Chronic kidney disease (CKD) and control rats were studied after 7 days of surgically induced unilateral plantaris muscle WO and a single leucine or saline load. In the basal state, 4E-BP1 phosphorylation was modestly depressed in non-WO muscle of CKD rats, whereas rpS6 phosphorylation was nearly completely suppressed. After oral leucine mTOR, S6K1 and rpS6 phosphorylation increased similarly in both groups, whereas the phospho-4E-BP1 response was modestly attenuated in CKD. WO alone activated the mTOR signaling pathway in control and CKD rats. In WO CKD, muscle leucine augmented mTOR and 4E-BP1 phosphorylation, but its effect on S6K1 phosphorylation was attenuated. Taken together, this study has established that the chronic uremic state impairs basal signaling through the mTOR anabolic pathway, an abnormality that may contribute to muscle wasting. However, despite this abnormality, leucine can stimulate this signaling pathway in CKD, although its effectiveness is partially attenuated, including in skeletal muscle undergoing sustained WO. Thus, although there is some resistance to leucine in CKD, the data suggest a potential role for leucine-rich supplements in the management of uremic muscle wasting.     

Formation and properties of flavoprotein-cytochrome hybrids by recombination of subunits from different species.

p-Cresol methylhydroxylases from four different pseudomonads differ in their isoelectric points and, to a lesser extent, in Mr values and substrate specificity. The enzymes from three species were isolated in homogeneous form, then resolved into their flavoprotein and cytochrome subunits, and the subunits were recombined to yield the nine possible hybrids (i.e. three intraspecies and six interspecies). The resulting flavocytochromes showed extensive similarities in steady-state kinetic parameters and in the dissociation constants of their subunits. Evidence is also presented that a fourth type of p-cresol methylhydroxylase, from Pseudomonas putida (N.C.I.B. 9869, form 'B'), the subunits of which cannot be isolated by the isoelectric focusing technique used to separate the subunits of the other flavocytochromes, nevertheless dissociates slowly at high dilution. The dissociation is reflected by a decline of catalytic activity with time. This process for the 'B' enzyme is prevented by the presence of substrate or an excess of a cytochrome subunit isolated from another enzyme species. Incubation of the dissociated subunits with p-cresol brings about extensive, albeit incomplete, re-association and regeneration of activity.     

Protection mechanisms of freely suspended animal cells (CRL 8018) from fluid-mechanical injury. Viscometric and bioreactor studies using serum, pluronic F68 and polyethylene glycol

We use bioreactor and viscometric studies to examine the mechanism by which three additives, fetal bovine serum (FBS), pluronic F68, and polyethylene glycol (PEG), protect the freely suspend CRL-8018 cells from damage due to interactions with bubbles in agitated bioreactors. In bioreactor studies, the protective effect of an addictive could be due to either changes in the ability of the cell resist shear (biological mechanism) or to changes in the medium properties that effect the level or frequency of forces experienced by the cells (physical mechanism). Bioreactor studies show that protection by all three addictives occurs whether the cells are grown in the presence of the addictives (long exposure) or the addictives are added to medium after the cells were exposed to detrimental agitation intensity (short exposure). In the viscometric studies, exposure of cells to laminar shear in the absence of gas-liquid interfaces assesses only the ability of the cells to resist a constant level of shear in a medium with or without an additive. Viscometric studies show that prolonged exposure to FBS makes the cells more shera tolerant, but that short (30-120 min) exposure to FBS does not affect their shear tolerance. We thus conclude that the protective effect of FBS in bioreactors id of both physical and biological nature. The biological contribution is metabolic in nature rather than fast acting. Viscometric studies show that either long or short exposure of the cells to either F68 or PEG does not make the cells more shear tolerant. WE therefore conclude that the protective effect of F68 and PEG does not make the cells more shear tolerant. We therefore conclude that the protective effect of F68 and PEG in bioreactors is physical in nature.     

Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel-plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.     

EFFECTS OF CURRENT VELOCITY AND LIGHT ENERGY ON THE STRUCTURE FO PERIPHYTON ASSEMBLAGE IN LABORATORY STREAMS1

Effects of current velocity and light energy on the taxonomica and physiognomic characteristics of periphyton assemblages were investigated in laboratory streams. The initial rate of colonization was related to current velocity, while the effects of light energy accounted for differences in species composition by the end of the experiment. Although the laboratory systems had many species in common during the realy stages of colonization, the experimental treatments generated differences in rates of communitydevelopment. synedra spp. were the early coloniters of the substrate, followed by an understory of Achnanthes spp. After day 16, Stigeoclonium tenue developed in the streams exposed to the higher photon flux density, but was rare in the shaded streams. The applicability of traditional successional theory to develoopmental patterns in lotic periphyton assemblages is discussed.     

AN INVESTIGATION OF DISTRIBUTIONAL PATTERNS IN THE DIATOM FLORA OF NETARTS BAY,OREGON, BY CORRESPONDENCE ANALYSIS1

Distributional patterns in assemblages of epiphytic and sediment-associated diatoms were investigated in Netarts Bay, Oregon. The method of reciprocal averaging revealed a floristic discontinuity between the epiphytic and sediment samples in ordination space. The basis for this discontinuity was the presence of a large number of sediment-associated taxa that were either very rare or not observed in the epiphytic samples. Within the sediment samples, the diatom flora formed a distributional continuum which had relatively high correlations with mean grain size, a sediment sorting coefficient, and the organic matter content of the sediment. A comparison of the flora in Netarts Bay with floras in other Oregon estuaries indicates that epiphytic, epilithic, and sediment-associated diatom assemblages do not exhibit conspicuous latitudinal changes along the coast of Oregon, and that many of the same taxa can be expected to occur in samples from comparable habitats in estuaries throughout the temperate regions of the world.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.