Location of the isoleucine arrest point in CHO and 3T3 cells

An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [³H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [³H] and [¹⁴C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.     

Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.     

Stage-specific localization of the small heat shock protein Hsp27 during oogenesis inDrosophila melanogaster

The developmental and heat shock-induced expression of the small heat shock protein Hsp27 was investigated by confocal microscopy of whole-mount immunostained preparations of ovarioles during oogenesis inDrosophila melanogaster. In unstressed flies, Hsp27 was mainly associated with germline nurse cells throughout egg development. A small group of somatic follicle cells also expressed Hsp27 specifically at stages 8 to 10 of oogenesis. Interestingly, this Hsp showed a different intracellular localization depending on the stages of egg chamber development. Thus Hsp27 was localized in the nucleus of nurse cells during the first stages of oogenesis (from germarium to stage 6) whereas it showed a perinuclear and cytoplasmic localization from stage 8. After a heat shock, Hsp27 accumulated in somatic follicle cells surrounding the egg chamber whereas the expression of this small Hsp did not seem to be enhanced in nurse cells. The stage-dependent pattern of intracellular localization of Hsp27 observed in nurse cells of unstressed flies was also observed following heat shock. At late stages of oogenesis, Hsp27 also showed a perinuclear distribution in follicle and nurse cells after heat stress. These observations suggest that different factors may modulate the expression and intracellular distribution of Hsp27. This modulation may be associated with the specific activities occurring in each particular cell type throughout oogenesis during both normal development and under heat shock conditions. Edited by: E.R. Schmidt     

Synergistic effects in theα 1- andβ 1-adrenergic regulations of intracellular calcium levels in striatal astrocytes

1. Using indo-1 as a calcium fluorescent probe, we have observed the following in striatal astrocytes in primary culture. 2. The stimulation of alpha-adrenoceptors induces a rapid rise in cytosolic calcium resulting from an internal calcium mobilization followed by an external calcium influx (4-min duration). 3. The stimulation of beta 1-adrenoceptors evokes only a slight internal calcium mobilization (90-sec duration). 4. The simultaneous stimulation of beta 1- and alpha 1-adrenoceptors induces a more prolonged calcium influx (10 min). The latter phenomenon could explain the calcium-dependent synergistic effects of alpha 1 and beta stimulation on cAMP production already described in the brain.     

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

Organization Environmental Footprint through Input‐Output Analysis: A Case Study in the Construction Sector

The implementation of global sustainability has gained worldwide attention in recent years. The Organization Environmental Footprint, which encompasses 14 impact categories, is a multicriteria measure of the environmental performance of goods and services provided by an organization from a life cycle perspective. In this article, the focus is on quantifying the Organization Environmental Footprint of a construction company in Spain. By applying an environmentally extended input‐output approach, its total footprint and impacts along the supply chain from two consecutive years were calculated. The results show that the environmental impacts from the second year of implementation were significantly higher than those from the first year. The impact category climate change was found to have experienced the greatest increase from one year to the other, with a 31% increase. This work provides an overview of 14 environmental impact categories of the company assessed, as well as recommendations for the implementation of this indicator in companies and public procurement. This approach could pave the way to shape organizations’ action plans and meet the European environmental challenges.     

Limited seed retention during winter inhibits vegetation establishment in spring,affecting lateral marsh expansion capacity

Coastal systems worldwide deliver vital ecosystem services, such as biodiversity, carbon sequestration, and coastal protection. Effectivity of these ecosystem services increases when vegetation is present. Understanding the mechanisms behind vegetation establishment in bio‐geomorphic systems is necessary to understand their ability to recover after erosive events and potential adaptations to climate change. In this study, we examined how seed availability affects vegetation establishment in the salt marsh–intertidal flat transition zone: the area with capacity for lateral marsh expansion. This requires vegetation establishment; therefore, seed availability is essential. In a 6‐month field experiment, we simulated a before and after winter seed dispersal at two locations, the salt‐marsh vegetation edge and the intertidal flat, and studied seed retention, the seed bank, and the seed viability of three pioneer marsh species: Salicornia procumbens, Aster tripolium, and Spartina anglica. During winter storm conditions, all supplied seeds eroded away with the sediment surface layer. After winter, supplied seeds from all three species were retained, mostly at the surface while 9% was bioturbated downwards. In the natural seed bank, A. tripolium and S. anglica were practically absent while S. procumbens occurred more frequently. The viability of S. procumbens seeds was highest at the surface, between 80% and 90%. The viability quickly decreased with depth, although viable S. procumbens seeds occurred up to 15 cm depth. Only when seeds were supplied after winter, many S. procumbens and some S. anglica individuals did establish successfully in the transition zone. Viable seed availability formed a vegetation establishment threshold, even with a local seed source. Our results suggest that, although boundary conditions such as elevation, inundation, and weather conditions were appropriate for vegetation establishment in spring, the soil surface in winter can be so dynamic that it limits lateral marsh expansion. These insights can be used for designing effective nature‐based coastal protection.