Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.     

BIOMASS AND DYNAMICS OF GROWTH OF ULVA SPECIES IN PALMONES RIVER ESTUARY1

During the last decade, the Palmones River estuary has undergone severe eutrophication followed by a green tide episode; two species of Ulva, rotundata Blid. and Ulva curvata (Kütz.) De Toni, were the main macroalgae responsible for this bloom. From November 1993 to December 1994, we followed the biomass, the growth dynamics, and tissue elemental composition (C:N:P)of Ulva species, as well as some physicochemical variables in the estuary. Maximum biomass (up to 375 g dry wt·m^?2 in some spots, corresponding to a thallus area index of nearly 17 m²Ulva·m^?2 sediment) were observed in June and December. However, the biomass varied among the sampling stations. Water nitrate, ammonia, and phosphate showed high concentrations throughout the year, with extremely high transient pulses, sustaining the high growth rates observed. Growth rates were estimated directly in the field. The rates were generally higher in Ulva discs maintained in net cages than those estimated by changes in biomass standing stock between two consecutive samplings. The difference between both estimates was used to quantify the importance of the processes causing loss of biomass, which were attributable to grazing, exported biomass, and thallus decomposition under anaerobic conditions resulting from extreme self-shading. Maximum chlorophyll content was found in winter, whereas the minimum was in spring. Atomic N:P ratios were generally higher in the algae than in the water. However, the absolute concentrations of tissue N and P were always higher than the critical levels for maximum growth, which suggests that growth was not limited by inorganic N or P availability. The results suggested that the increase in nutrient loading in the river may have triggered the massive development of green algae and that light limitation and temperature stress in summer seem to be the main factors controlling the abundance of Ulva in the estuary. In addition to light availability and thermal stress, the different loss processes may have a decisive role in the dynamics of Ulva biomass.     

Inositol 1,4,5-triphosphate phosphatase activity in membranes isolated from amphibian skeletal muscle [corrected

The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.     

Forest fragmentation and rhinocryptid nest predation in central Chile

Fragmentation of forest landscapes can raise the intensity of nest predation by increasing the abundance and richness of generalist or introduced predators. Understory foraging birds, such as rhinocryptids, can be highly vulnerable to nest predation in fragmented landscapes because they often place their nests on the ground. Temperate deciduous forests in Chile have been intensively fragmented in the last centuries, causing changes in nest predator densities. We tested if predation of artificial nests, mimicking those of rhinocryptids, placed on and above ground was higher in the remnant fragments of central Chile due to an increase in predator abundance. The rate of nest predation in forest remnants was larger than in native continuous forest. Small mammals were the main nest predators. Despite high predation rates, the abundance of rhinocryptids is higher in forest remnants, suggesting that fragments might constitute ecological traps.     

Double-strand break toxicity is chromatin context independent

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.     

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998