A radial mode ultrasonic horn for the inactivation of <i>Escherichia coli</i> K12

Tuned cylindrical radial mode ultrasonic horns offer advantages over ultrasonic probes in the design of flow-through devices for bacterial inactivation. This study presents a comparison of the effectiveness of a radial horn and probe in the inactivation of Escherichia coli K12. The radial horn is designed using finite element analysis and the predicted modal parameters are validated using experimental modal analysis. A validated finite element model of the probe is also presented. Visual studies of the cavitation fields produced by the radial horn and probe are carried out using luminol and also backlighting to demonstrate the advantages of radial horns in producing a more focused cavitation field with widely dispersed streamers. Microbiological studies show that, for the same power density, better inactivation of E. coli K12 is achieved using the radial horn and, also, the radial horn offers greater achievable power density resulting in further improvements in bacterial inactivation. The radial horn is shown to be more effective than the probe device and offers opportunities to design in-line flow-through devices for processing applications

Problem-based Learning: A Critical Rationalist Perspective

Although problem-based learning is being adopted by many institutions around the world as an effective model of learning in higher education, there is a surprising lack of critique in the problem-based learning literature in relation to its philosophical characteristics. This paper explores epistemology as a starting point for investigating the theoretical underpinnings of problem-based learning as a learning model. Criticisms of empiricism are analysed in terms of the perceived learning outcomes of learners undertaking a problem-based learning curriculum. It is argued that models of empiricism theorised by philosophers such as Bacon, Locke and Hume cannot fully account for the learning model found in problem-based learning curricula. It is proposed that an alternative epistemological approach is needed. The work of Karl Popper is discussed, whose critical rationalist epistemology emphasises the generation of bold conjectures and criticism. Popper's work shows a positive contribution to the demands of higher education, characterised by learners who are serious about making professional progress. The paper concludes by critically analysing the tensions and contradictions of problem-based learning in light of Popper's epistemological theory of critical rationalism. It is argued that a critical rationalist perspective has educational benefits for students as it creates an environment rich in critical thinking, reading and writing and values disjunction and challenge

Axonal and dendritic endocytic pathways in cultured neurons

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport-t of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially

Regulated internalization of caveolae

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network

Transcytosis in MDCK cells: identification of glycoproteins transported bidirectionally between both plasma membrane domains.

MDCK cells display fluid-phase transcytosis in both directions across the cell. Transcytosis of cell surface molecules was estimated by electron microscopic analysis of streptavidin-gold-labeled frozen sections of biotinylated cells. Within 3 h, approximately 10% of the surface molecules, biotinylated on the starting membrane domain, were detected on the opposite surface domain irrespective of the direction of transcytosis. This suggests that the transcytosis rates for surface molecules are equal in both directions across the cell as shown previously for fluid-phase markers

Nocodazole-Dependent Transport, and Brefeldin A-Sensitive Processing and Sorting, of Newly Synthesized Membrane-Proteins in Cultured Neurons

The envelope glycoproteins of Semliki Forest virus (SFV), Vesicular Stomatitis virus (VSV), and Influenza Fowl Plague virus (FPV) are vectorially targeted in neurons to the plasma membrane of dendrites (SFV and VSV) and axons (FPV), To gain insight into the mechanisms responsible for such polarized delivery we have examined the effects on neurons of nocodazole and brefeldin A (BFA), which are known to cause microtubule depolymerization and disassembly of the Golgi apparatus, respectively, Nocodazole treatment blocked transport of all viral glycoproteins to both axons and dendrites, BFA treatment induced disruption of the Golgi complex, including the trans-Golgi network (TGN), and tubulation of endosomes, However, the delivery of the SFV and FPV glycoproteins to the cell surface was not affected significantly by BFA, although processing and sorting were altered as revealed by surface biotinylation and immunofluorescence microscopy of fixed nonpermeabilized cells, These results demonstrate the involvement of microtubules in axonal and dendritic transport of integral membrane glycoproteins, and the existence of a BFA-sensitive component in the sorting but not in the transport machinery

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer to the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Videomicroscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I, Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney, Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb, is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb, plays a role in vesicular transport to the apical plasma membrane in MDCK cells