Calcium pools,calcium entry,and cell growth

The Ca²⁺ pump and Ca²⁺ release functions of intracellular Ca²⁺ pools have been well characterized. However, the nature and identity of Ca²⁺ pools as well as the physiological implications of Ca²⁺levels within them, have remained elusive. Ca²⁺ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca²⁺ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca²⁺ pools, the levels of Ca²⁺ within Ca²⁺ pools, and how these intraluminal Ca²⁺ levels may be physiologically related to ER function. Experiments utilizingin situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluroescent Ca²⁺ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ pools within cells, and large changes in the levels of Ca²⁺ within pools following InsP₃-mediated Ca²⁺ release. Such changes in Ca²⁺ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca²⁺-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca²⁺ changes with respect to cell growth. Emptying of pools using Ca²⁺ pump blockers can result in cells entering a stable quiescent G₀-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca²⁺ pools return, and cells reenter the cell cycle. During the Ca²⁺ pool-depleted growth-arrested state, cells express a Ca²⁺ influx channel that is distinct from the store-operated Ca²⁺ influx channels activated after short-term depletion of Ca²⁺ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca²⁺ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.     

Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

Proteolipid/DM-20 proteins bearing the paralytic tremor mutation in peripheral nerves and transfected Cos-7 cells

Paralytic tremor (Plp-pt) is a missense mutation of the myelin proteolipid gene (Plp) in rabbits. The myelin yield in the Plp-pt brain is reduced and the protein and lipid composition of central nervous system (CNS) myelin is abnormal. We studied the intracellular transport of the normal and Plp-pt mutant PLP and DM-20 in transiently transfected Cos-7 cells. While the mutant PLP accumulates in the rough endoplasmic reticulum and does not reach the plasma membrane, the spliced isoform of PLP, mutant DM-20, is normally transported to the cell surface and integrated into the membrane. Analysis of rabbit sciatic nerves revealed that concentration of peripheral nervous system (PNS) myelin proteins is normal in Plp-pt myelin. In the PNS like in the CNS, the level of Plp gene products is subnormal. But this does not affect myelination, in the PNS where PLP, present in low concentration, is not a structural component of compact myelin. The normal level of Plp gene expression in Schwann cells is low and these results suggest that, in the Plp-pt PNS, Schwann cell function is not affected by the deficiency in PLP and/or the impairment of intracellular PLP transport. Special issue dedicated to Dr Marion E. Smith.     

Disentangling natural and anthropogenic drivers of changes in a shallow lake using palaeolimnology and historical archives

Shallow lakes are susceptible to catastrophic regime shifts characterised by the presence or absence or macrophytes. However, the long-term controls on macrophyte succession in shallow lakes are incompletely understood. To investigate this, we analysed multiple sediment proxies in Lake Rotcze (Eastern Poland), a small, shallow and densely macrophyte-covered lake to (1) reconstruct the ‘reference conditions’ (sensu WFD) and development of the lake in recent centuries, (2) compare historical evidence with the sedimentary record, and (3) identify the natural and anthropogenic drivers of macrophyte succession. Before the twentieth century, conditions in the lake may be referred to as ‘reference conditions’. Subsequently forest clearance in the catchment resulted in lower water transparency, but concurrent catchment drainage lowered water levels and increased macrophyte development. Since 1950 elevated nutrient supply and climatically driven increases in water levels led to the deterioration of water transparency and partial macrophyte withdrawal. At the end of the twentieth century lake-level drawdown led to low phytoplankton biomass and clear water creating a novel ecosystem where macrophytes invade the whole lake. These patterns suggest that both natural and anthropogenically induced water level fluctuations have been critical drivers of macrophyte development.     

A study on calcium oxalate crystals in <Emphasis Type="Italic">Tinantia anomala</Emphasis> (Commelinaceae) with special reference to ultrastructural changes during anther development

Calcium oxalate (CaOx) crystals in higher plants occur in five forms: raphides, styloids, prisms, druses, and crystal sand. CaOx crystals are formed in almost all tissues in intravacuolar crystal chambers. However, the mechanism of crystallization and the role of CaOx crystals have not been clearly explained. The aim of this study was to explore the occurrence and location of CaOx crystals in organs of Tinantia anomala (Torr.) C.B. Clarke (Commelinaceae) with special attention to ultrastructural changes in the quantity of tapetal raphides during microsporogenesis. We observed various parts of the plant, that is, stems, leaves, sepals, petals, anthers, staminal trichomes and stigmatic papillae and identified CaOx crystals in all parts except staminal trichomes and stigmatic papillae in Tinantia anomala. Three morphological forms: styloids, raphides and prisms were found in different amounts in different parts of the plant. Furthermore, in this species, we identified tapetal raphides in anthers. The number of tapetal raphides changed during microsporogenesis. At the beginning of meiosis, the biosynthesis of raphides proceeded intensively in the provacuoles. These organelles were formed from the endoplasmic reticulum system. In the tetrad stage, we observed vacuoles with needle-shaped raphides (type I) always localised in the centre of the organelle. When the amoeboid tapetum was degenerating, vacuoles also began to fade. We observed a small number of raphides in the stage of mature pollen grains.     

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved, e.g., through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems, such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots, including physical and chemical ones, such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates, or media [reviewed by Ragonezi et al. (Trees 24(6):975–992, 2010)]. The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant-growth promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also to help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme

Summary The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants. The 15K lysozyme was also found to be expressed in E. coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25. These observations indicate that the 15K lysozyme is the gene 25 product.