Dissemination of spores in a glasshouse: Pattern or chaos?

The dissemination of particles in a glasshouse with three united compartments was observed from experiments withLycopodium sp. The experiments were performed in a glasshouse with open and closed ventilation openings, spore sources on two heights above soil surface, and in two consecutive time intervals. The spores were caught with self constructed spore traps placed at regular distances from each other. Air circulation was observed using smoke-puffs. High ventilation caused a rapid cleaning of the air while in a closed glasshouse spores remained suspended for quite a long time. Though the three compartments of the glasshouse were not separated by walls, a so-called cell structure of the individual compartments could be indicated.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

Cryptococcus neoformans and its cell wall components induce similar cytokine profiles in human peripheral blood mononuclear cells despite differences in structure

We studied the cytokine profile of peripheral blood mononuclear cells after stimulation with various cryptococcal strains or its purified cell wall components. After 3 h of stimulation, tumor necrosis factor (TNF) alpha levels were strongly increased, whereas interferon (IFN) gamma and interleukin (IL) 10 levels were increased only slightly, or not at all (respectively). In contrast, after 18 h, TNF-alpha and IFN-gamma levels were (strongly) decreased, whereas the IL-10 levels were increased. The IL-1beta, IL-6 and IL-8 levels were equally high throughout the experiment. In order to establish which of the cryptococcal envelope components contributed most to the observed cytokine profile induced by whole cryptococci, glucuronoxylomannan, galactoxylomannan and mannoproteins were purified and partially characterized biochemically. All cryptococcal components elicited a similar cytokine pattern despite the differences in structure.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

Enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway

Combinations of lactococcal strains from various origins with divers properties were developed as new starters for new dairy products. Flavour formation by such tailor-made cultures was studied. In some cases, a strongly enhanced flavour was observed. For instance, the combination of B1157 and SK110 strains in milk resulted in a very strong chocolate-like flavour. B1157 produces only a moderate chocolate-like flavour, whereas SK110 alone fails to produce this flavour. Headspace gas chromatography results corroborate the organoleptic evaluations. High levels of branched-chain aldehydes were found when B1157 and SK110 were grown together. The enzyme activities involved in this pathway were studied; both strains contain transaminase activity. Although B1157 had a very high amino acid decarboxylating activity, its release of amino acids from milk protein was limited. SK110 was strongly limited in decarboxylating activity, although this strain is very active in proteolysis. By combining these strains, the substrates released by SK110 can directly be used by the other strain, resulting in the completion of the whole flavour-formation pathway. This opens new avenues for the preparation of tailor-made cultures.     

Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan

N,N,N-Trimethylated chitosan (TMC) is a biodegradable polymer emerging as a promising nonviral vector for nucleic acid and protein delivery. In the present study, we investigated whether the introduction of thiol groups in TMC enhances the extracellular stability of the complexes based on this polymer and promotes the intracellular release of siRNA. The gene silencing activity and the cellular cytotoxicity of polyplexes based on thiolated TMC were compared with those based on the nonthiolated counterpart and the regularly used lipidic transfection agent Lipofectamine. Incubation of H1299 human lung cancer cells expressing firefly luciferase with siRNA/thiolated TMC polyplexes resulted in 60-80% gene silencing activity, whereas complexes based on nonthiolated TMC showed less silencing (40%). The silencing activity of the complexes based on Lipofectamine 2000 was about 60-70%. Importantly, the TMC-SH polyplexes retained their silencing activity in the presence of hyaluronic acid, while nonthiolated TMC polyplexes hardly showed any silencing activity, demonstrating their stability against competing anionic macromolecules. Under the experimental conditions tested, the cytotoxicity of the thiolated and nonthiolated siRNA complexes was lower than those based on Lipofectamine. Given the good extracellular stability and good silencing activity, it is concluded that polyplexes based on TMC-SH are attractive systems for further in vivo evaluations.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.