Subnucleolar location of fibrillarin and variation in its levels during the cell cycle and during differentiation of plant cells

The nucleolar protein fibrillarin has been studied in onion cells; it is detected as an Mr 37,000 protein by immunoblotting using a human autoimmune serum. Quantitative immunoelectron microscopy showed that most fibrillarin is localized in the transition zone between the fibrillar center (FC) and the dense fibrillar component (DFC) as well as in the priximal zone of the DFC, where the labeling shows a gradual decrease out-ward until it reaches insignificant levels in the distal zone of the DFC. Thus, fibrillarin is not uniformly distributed throughout the DFC of plant cells. This result supports the hypothesis that the morphologically homogeneous DFC may not be uniform in function; it is also in agreement with the hypothesized vectorial flow of ribosome biogenesis through the same compartments. Data are also presented showing that the amount of fibrillarin increase when nucleolar activity increases in G2, and probably decreases when nucleolar activity decreases during differentiation.     

Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A₁₂) and globular hydrophilic and amphiphilic (G^H₄, G^A₄, G^A₂, and G^A₁) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G^H₄ and G^A₄ decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A₁₂ AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G^A₄ AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G^A₄ forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.     

The biosynthesis of glucose containing insect lipid linked oligosaccharide and its possible role in glycoprotein assembly

Summary Microsome enriched Ceratitis capitata extracts synthesized a glucosylated lipid linked oligosaccharide. Its properties were closely related to those of the previously described insect mannosylated dolichyl diphosphate oligosaccharides and almost the same as those of the rat liver dolichyl-diphosphate-(GlcNAc)₂-(Man)₉-(Glc)_1–3. The saccharide moiety of, the latter was transferred to an unknown endogenous protein-like acceptor by the fly extracts. These represent the first evidence of a protein glycosylation in a pluricellular invertebrate through dolichyl derivatives.Abbreviations Dol-P dolichyl phosphate - Dol-P-P dolichyl diphosphate     

Fibroblastlike primary cells from human colon adenocarcinoma explants: Collagen biosynthesis

Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology; b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells. Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent phenotypic alteration in the tumor-associated fibroblastlike cells.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Effects of eel (Anguilla anguilla L.) removals from selected sites of a stream on its subsequent densities

We assessed the effect of eel (Anguilla anguilla) removal from three sites of a Cantabrian stream upon its subsequent densities. In the first sample (Sept. 1986) numbers and densities were estimated as 43, 45 and 84 ind and 3490, 3030 and 3750 ind ha ⁻¹. Removal of these eels reduced the subsequent numbers and densities which, except on two occasions, were never reached again during the two years (eleven estimates) of study. Highest densities were recorded in the uppermost site in May and July, 1987, coincident with a strong drought and the lowest densities occurred in 1988 during a normal wet year. We hypothesize first that, because of a selective underground homing behaviour of eels, electro-fishing is inefficient and results in underestimates of the population. Second, seasonal variations of water discharge and droughts may not influence the homing behaviour of'eels until a threshold of dryness is reached. If this occurs, eels abandon their refuges and move towards the stream bottom. It seems that in Arroyo Chabatchos this threshold was exceeded in the summer of 1987 when the highest densities were estimated. The re-colonization of these sites experimentally depleted of eels, is a slow procces that lasts for, at least, two years.     

Time-dependent regulation of muscle caveolin activation and insulin signalling in response to high-fat diet

We studied the effect of high-fat diet on the expression and activation of the three caveolins in rat skeletal muscle and their association with the insulin signalling cascade. Initial response was characterized by increased signalling through Cav-1 and Cav-3 phosphorylation, suggesting that both participate in an initial acute response to the calorie surplus. Afterwards, Cav-1 signalling was slightly reduced, whereas Cav-3 remained active. Late chronic phase signalling through both proteins was impaired inducing a prediabetic state. Summarizing, caveolins seem to mediate a time-dependent regulation of insulin cascade in response to high-fat diet in muscle.     

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.     

Tunable Stochastic Pulsing in the Escherichia coli Multiple Antibiotic Resistance Network from Interlinked Positive and Negative Feedback Loops

Cells live in uncertain, dynamic environments and have many mechanisms for sensing and responding to changes in their surroundings. However, sudden fluctuations in the environment can be catastrophic to a population if it relies solely on sensory responses, which have a delay associated with them. Cells can reconcile these effects by using a tunable stochastic response, where in the absence of a stressor they create phenotypic diversity within an isogenic population, but use a deterministic response when stressors are sensed. Here, we develop a stochastic model of the multiple antibiotic resistance network of Escherichia coli and show that it can produce tunable stochastic pulses in the activator MarA. In particular, we show that a combination of interlinked positive and negative feedback loops plays an important role in setting the dynamics of the stochastic pulses. Negative feedback produces a pulsatile response that is tunable, while positive feedback serves to amplify the effect. Our simulations show that the uninduced native network is in a parameter regime that is of low cost to the cell (taxing resistance mechanisms are expressed infrequently) and also elevated noise strength (phenotypic variability is high). The stochastic pulsing can be tuned by MarA induction such that variability is decreased once stresses are sensed, avoiding the detrimental effects of noise when an optimal MarA concentration is needed. We further show that variability in the expression of MarA can act as a bet hedging mechanism, allowing for survival in time-varying stress environments, however this effect is tunable to allow for a fully induced, deterministic response in the presence of a stressor.