Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.     

Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide

Abstract Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa . This study may help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.     

High resolution scanning electron micrographic study of dissociated mouse taste cells

New techniques for enzymatic dissociation of mammalian tastecells allowed us to study, for the first time, the morphologyof murine taste receptor cells using high resolution scanningelectron microscopy. Cell shape varied from spindle to bipolarto lamellar, similar to shapes previously described in cellsfrom amphibian taste buds. Cell length varied from 19 to 65µm (39 ± 19 µm), with width averaging 6 ±3.4 µm. A rare picture of the apical microvilli of a tastereceptor cell, and a view of microvilli within a taste pore,suggest that at any given time, five to eight taste cells maybe exposed to the oral cavity. Assuming a cell life-span of10 days, and 50 cells per bud, all of which eventually reachthe taste pore, one can calculate that the average cell is exposedto the oral environment for     

A split-pot experiment with sorghum to test a root water uptake partitioning model

Correct modeling of root water uptake partitioning over depth is an important issue in hydrological and crop growth models. Recently a physically based model to describe root water uptake was developed at single root scale and upscaled to the root system scale considering a homogeneous distribution of roots per soil layer. Root water uptake partitioning is calculated over soil layers or compartments as a function of respective soil hydraulic conditions, specifically the soil matric flux potential, root characteristics and a root system efficiency factor to compensate for within-layer root system heterogeneities. The performance of this model was tested in an experiment performed in two-compartment split-pot lysimeters with sorghum plants. The compartments were submitted to different irrigation cycles resulting in contrasting water contents over time. The root system efficiency factor was determined to be about 0.05. Release of water from roots to soil was predicted and observed on several occasions during the experiment; however, model predictions suggested root water release to occur more often and at a higher rate than observed. This may be due to not considering internal root system resistances, thus overestimating the ease with which roots can act as conductors of water. Excluding these erroneous predictions from the dataset, statistical indices show model performance to be of good quality.     

The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans

Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.     

Construction of first-generation lactococcal integrative cloning vectors

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (ery ^r) gene obtained from L. lactis IL1837. Integration of the ery ^r gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nis ^r) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nis ^r fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for >200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (Nis ^r ) transformant suggested that the integrated nis ^r gene may be amplifying within the host chromosome. Correspondence to: S. K. Harlander