RNA interference in the moss Physcomitrella patens

The moss Physcomitrella patens performs efficient homologous recombination, which allows for the study of individual gene function by generating gene disruptions. Yet, if the gene of study is essential, gene disruptions cannot be isolated in the predominantly haploid P. patens. Additionally, disruption of a gene does not always generate observable phenotypes due to redundant functions from related genes. However, RNA interference (RNAi) can provide mutants for both of these situations. We show that RNAi disrupts gene expression in P. patens, adding a significant tool for the study of plant gene function. To assay for RNAi in moss, we constructed a line (NLS-4) expressing a nuclearly localized green fluorescent protein (GFP):beta-glucuronidase (GUS) fusion reporter protein. We targeted the reporter protein with two RNAi constructs, GUS-RNAi and GFP-RNAi, expressed transiently by particle bombardment. Transformed protonemal cells are marked by cobombardment with dsRed2, which diffuses between the nucleus and cytoplasm. Cells transformed with control constructs have nuclear/cytoplasmic red fluorescence and nuclear green fluorescence. In cells transformed with GUS-RNAi or GFP-RNAi constructs, the nuclear green fluorescence was reduced on average 9-fold as soon as 48 h after transformation. Moreover, isolated lines of NLS-4 stably transformed with GUS-RNAi construct have silenced nuclear GFP, indicating that RNAi is propagated stably. Thus, RNAi adds a powerful tool for functional analysis of plant genes in moss.     

Influence of air-ions on selected microorganisms

In the following work the influence of air-ions and ozone on the selected microorganisms, first of all on Staphylococcus epidermidis is discussed. Carried out investigations proved that the levels of air-ions concentrations, causing specific changes in the development of particular microorganisms, are very differentiated and depend on individual parameters of those organisms. These levels are generally characterized by high values of concentrations, range n +/- =4 x 10(6) ions/cm3 and are connected with the type of the air-ions used. However investigations did not confirm any negative influence of the positive air-ions seen as a destructive factor upon any of the investigated microorganisms.     

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.     

Demonstration of nerve fibers immunoreactive to peptide N-terminal histidine C-terminal isoleucine (PHI) and vasoactive intestinal peptide (VIP) in the pig pineal gland

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.     

Molecular simulation study of cooperativity in hydrophobic association: clusters of four hydrophobic particles

The multibody contribution to the potential of mean force (PMF) of hydrophobic association of four methane molecules in water was investigated by means of umbrella-sampling molecular dynamics. Two systems were considered: (i). a trigonal pyramid with three methane molecules at contact distance forming a fixed base, the fourth molecule being placed on the top with variable distance from the base; and (ii). a regular uniformly expanding tetrahedron. Methane-methane distances as far as 12.5 A, i.e. beyond the second solvent-separated minimum of the PMF, were considered to address the baseline problem. In contrast to the small effect in the three-body case studied previously (Protein Sci 9 (2000) 1235), the multibody contribution was found to amount to approximately 0.2 kcal/mol per methane-methane pair, or approximately 25% of the depth of the contact minimum in the PMF. The main effect of the multibody contribution to the PMF is a reduction of the height of the barrier between the contact and solvent separated minima and a narrowing of the region of its maximum, while the region of the contact minimum is affected only weakly. The reduction of the barrier is due to four-body contributions. The cooperative contributions to the PMF agree very well with those computed from the molecular surface of the systems under consideration, which further supports earlier observations that the molecular surface can be used with good accuracy to describe the energetics of hydrophobic association.     

Biological activity of pentachlorophenol on the digestive gland cells of the freshwater mussel Unio tumidus

Many chlorinated phenols and their derivatives are used extensively as insecticides, fungicides and herbicides by industrial and agricultural users throughout the world. Among these substances, pentachlorophenol (PCP) is a broad-spectrum biocide, which is still used as a wood preservative. In this paper, the digestive gland cells were used to assess the effect of PCP in the range of concentrations 3.75-75 microM (0.01-0.2 ppm) on oxidative DNA damage, fluidity changes and peroxidation activity in the plasma membrane. The toxic property of PCP on DNA strand breakage was studied using the comet assay. The results showed that pentachlorophenol in the range of 37.5-75 microM contributed to these lesions. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric method using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS). It was shown that PC did not influence the surface of plasma membrane but contributed to the increase in the fluidity of the internal region of the lipid bilayer in the range of concentrations 18.75-75 microM (0.05-0.2 ppm). We also examined the effect of PCP on the lipid peroxidation. To imply its peroxidation properties the spectrophotometry method was used to measure the level of malondialdehyde (MDA), one of the endpoints of the peroxidation of polyunsaturated fatty acids. The obtained results showed that PCP in the used doses did not initiate the formation of lipid peroxides. Thus, our investigation indicates that PCP can behave as a prooxidant agent but its action depends on the used doses and parameters chosen for the research.     

The effects of insulin and β-adrenergic stimulation on glucose transport,glut 4 and PKB activation in the myocardium of lean and obese non-insulin dependent diabetes mellitus rats

Glucose uptake, glut 4 translocation and activation of protein kinase B were measured in Langendorff perfused hearts from (i) Wistar control, (ii) lean, neonatal Streptozotocin induced (Stz) and (iii) Zucker (fa/fa) obese diabetic rats of 10–12 weeks old. Hearts were subjected to stimulation with insulin, isoproterenol (-adrenergic agonist) or a combination of insulin and isoproterenol, during the perfusion protocol. Basal myocardial glucose uptake was impaired in both diabetic models, but could be stimulated significantly by insulin. In the Zucker rats, the time-course of insulin action was delayed. Insulin and -stimulation of glucose uptake were not additive. Evaluation of sarcolemmal membranes from these hearts showed that the affinity of glut 4 was significantly lower in the Zucker but not in the Stz hearts while a reduced affinity found with a combination of insulin and -stimulation in control hearts, was absent in both diabetic models. Total membrane lysates were analyzed for glut 4 expression while an intracellular component was generated to quantify translocation on stimulation as well as activity of protein kinase B (PKB). At this age, the neonatal Streptozotocin induced diabetic animals presented with more faulty regulation concerning adrenergic stimulated effects on elements of this signal transduction pathway while the Zucker fa/fa animals showed larger deviations in insulin stimulated effects. The overall response of the Zucker myocardium was poorer than that of the Stz group. No significant modulation of -adrenergic signaling on insulin stimulated glucose uptake was found. The PI-3-kinase inhibitor wortmannin, could abolish glucose uptake as well as PKB activation elicited by both insulin and isoproterenol.     

Clonal Analysis Provides Evidence for Transient Initial Cells in Shoot Apical Meristems of Seed Plants

Abstract Drift of mutated sectors in sectorial or mericlinal plant chimeras has been interpreted as indirect evidence of initial impermanence at the apex. However, the same effect may result from mutation in noninitial cells positioned close to the vertex of the apical dome. Clonal analysis of the cell packets present in the superficial layer of spruce and magnolia apices provided the library of patterns suggesting that the position and the number of initial cells, and in some cases also the meristem axis inclination, may change over time. Multicellular clones originating from a single cell have been found in the geometric center of some apices, whereas in other apices the cellular center (where three or four clonal borders meet) did not correspond to the geometric center of the apex. Such effects may result only from initial impermanence.