Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Effect of various growth-promoting factors on preimplantation bovine embryo development in vitro

One-cell bovine embryos produced by in vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were cultured in chemically defined medium (CDM) to which the following growth-promoting factors were added separately: transferrin (Tf, 10 ug/ml), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-betal (TGF-beta1), insulin-like growth factor-one (IGF-I), insulin-like growth factor-two (IGF-II), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), all at 10 ng/ml. The embryos were cultured for a total of 10 days and were assessed. The culture medium was changed every 2 days. There were no beneficial effects of growth factors on embryo development to morula or hatched blastocyst stages of development. However, supplementation with EGF approached significance (P<0.08) when compared with that of CDM alone at the blastocyst stage of development. The results suggest that supplementation with growth factors does not improve bovine IVM-IVF embryo development when cultured under chemically-defined conditions.     

Some aspects of the infrastructure of the ‘Stäbchendrüscnzellen’, a peculiar cell associated with the endothelium of the bulbus arteriosus and with other fish tissues*

The fine structure of an unusual cell, the ‘Stäbchendriisenzellen’ (the so-called foliaceous or rodlet cells of several authors), associated with the endothelium of the vascular system of marine and freshwater fishes was studied in the goldfish Carassius auratus (L.). The bulbus arteriosus was fixed with either a 6 % solution of buffered glutaraldehyde or with one part of a 3 % solution of lanthanum nitrate plus two parts of a 3 % solution of buffered glutaraldehyde and then post-fixed in a 1.2% solution of osmium tetroxide. Electron micrographs of the vascular tissue show four cell forms which appear to be phases in the life cycle of the endothelium-associated cell. Two of the phases are not encased and are characterized by their inclusions: in one cell these are crystalline, while in the second, they are amorphous and granular. In the other phases the cell is encased partially or completely within an apparently contractile fibrous wall surrounded by the plasmalemma. In the encased phases, the arrangement and condition of the cell organelles appears to have been changed, and in the fully-encased phase mitochondrial activity seems to have decreased. This apparent change in the mitochondrial activity is accompanied by a thickening of the mitochondrial membrane from 48 to 109 Å. The micrographs seem to indicate that this cell in question behaves as a foreign body and, in some way, may be interacting with the epithelial tissue of the bulbus arteriosus.     

Auxin-induced cell expansion of potato tuber and chicory root: Role of hydroxyproline synthesis

We have reinvestigated the role of protein-bound hydroxyproline (extensin) in auxininduced cell enlargement using discs excised from tubers of Solanum tuberosum L. cv. Pentland Crown and from roots of Cichorium intybus L. cv. Magdeburg. Extensin increases markedly in potato tuber discs treated with water and auxin, and the hydroxyproline is primarily in the cell wall. 2,2'-Dipyridyl totally inhibits both hydroxyproline synthesis and auxin-induced cell expansion in potato with the inhibitions being reversed in parallel by Fe²⁺. Free hydroxyproline also totally prevents induced cell enlargement. Pretreatment with gibberellic acid totally inhibits subsequent auxin-induced cell expansion but does not inhibit hydroxyproline synthesis. Therefore, the level of hydroxyproline does not control auxin-induced cell enlargement in potato tuber discs. Other interpretations are discussed but we conclude that extensin biosynthesis is necessary for auxin-dependent cell expansion as inhibition of the synthesis prevents the induced expansion. Dipyridyl and free hydroxyproline partially inhibit auxin-induced cell enlargement in chicory root discs. Thus a component of the auxin-dependent cell enlargement in chicory is also dependent on extensin synthesis.     

The ultrastructure and vascular supply of the different fibre types in the axial muscle of the sturgeon Acipenser stellatus,Pallas

Summary The ultrastructure and vascular supply of the different fibre types in the lateral muscles of the sturgeon Acipenser stellatus were studied by light- and electron microscopy and morphometry. Three fibre types form separate layers without intermingling. The red fibres are superficial, the white fibres deep and the intermediate fibres between them. From morphometric analyses, the mitochondrial volume fraction in red fibres is 30%, in intermediate fibres 3.7% and in white fibres 0.7%. Z lines are most fuzzy in the red fibres. Triads of the sarcotubular system are always situated at the level of the Z discs. In red fibres the three elements are arranged in a series along the myofibrils, whereas in white fibres they are arranged transversely and in the intermediate fibres they are aligned obliquely. The number of capillaries surrounding each fibre is 2.3, 0.9 and 0.2 for the red, intermediate and white fibres, respectively. In red fibres 16% of the surface is directly covered by capillaries. The corresponding percentages for intermediate and white fibres are 5 and 1, respectively. Per unit volume of the fibre, the directly vascularised fibre surface in red fibres is about ten times larger than that of white fibres.The degree of vascularisation of the fibre types is directly related to the volume fraction of mitochondria, and thus to their aerobic capacities.     

2,4-Dichlorophenoxyacetic acid and the de novo synthesis of invertase in chicory root tissue

Using a dual radioactive labelling technique, the large 2,4-D induced increase in invertase activity in root tissue of chicory (Cichorium intybus) could not be attributed to de novo protein synthesis. The highly active enzyme could have arisen by modification of an inactive enzyme precursor.     

Vasoactive intestinal peptide (VIP): an amnestic neuropeptide

Vasoactive intestinal peptide (VIP) is a neuropeptide present in high concentrations in the hippocampus. The studies reported here demonstrate that VIP administered into the third ventricle of the brain caused amnesia in mice trained on a left-right footshock avoidance task in a T-maze. VIP resulted in amnesia when administered directly into the rostral portion of the hippocampus at a 10-fold lower dose than was needed to produce amnesia when VIP was administered intracerebroventricularly. When VIP was administered 24 hr after training, it failed to impair retention measured a week later. VIP receptor antagonist ([4-Cl-D-Phe6,Leu17]VIP) enhanced retention when administered into the rostral portion of the hippocampus, suggesting that VIP plays a physiological role in memory modulation. VIP receptor antagonist administered 24 hr after training did not facilitate retention. To gain some insight as to how VIP may be affecting memory processing, we determined if some memory-improving compounds showed a selective ability to block amnesia induced by VIP. The amnestic effect of VIP was blocked by peripheral administration of the memory-enhancing agents, arecoline, naloxone and ST 587 (a noradrenergic receptor agonist) but not by cholecystokinin octapeptide. Central administration of arecoline, but not neuropeptide Y, blocked the amnestic effect of VIP. It is concluded that VIP is a potent amnestic peptide.     

Abrupt burial imparts persistent changes to the bacterial diversity of turbidite‐associated sediment profiles

The emplacement of subaqueous gravity‐driven sediment flows imposes a significant physical and geochemical impact on underlying sediment and microbial communities. Although previous studies have established lasting mineralogical and biological signatures of turbidite deposition, the response of bacteria and archaea within and beneath debris flows remains poorly constrained. Both bacterial cells associated with the underlying sediment and those attached to allochthonous material must respond to substantially altered environmental conditions and selective pressures. As a consequence, turbidites and underlying sediments provide an exceptional opportunity to examine (i) the microbial community response to rapid sedimentation and (ii) the preservation and identification of displaced micro‐organisms. We collected Illumina MiSeq sequence libraries across turbidite boundaries at ~26 cm sediment depth in La Jolla Canyon off the coast of California, and at ~50 cm depth in meromictic Twin Lake, Hennepin County, MN. 16S rRNA gene signatures of relict and active bacterial populations exhibit persistent differences attributable to turbidite deposition. In particular, both the marine and lacustrine turbidite boundaries are sharply demarcated by the abundance and diversity of Chloroflexi, suggesting a characteristic sensitivity to sediment disturbance history or to differences in organic substrates across turbidite profiles. Variations in the abundance of putative dissimilatory sulfate‐reducing Deltaproteobacteria across the buried La Jolla Canyon sediment–water interface reflect turbidite‐induced changes to the geochemical environment. Species‐level distinctions within the Deltaproteobacteria clearly conform to the sedimentological boundary, suggesting a continuing impact of genetic inheritance distinguishable from broader trends attributable to selective pressure. Abrupt, <1‐cm scale changes in bacterial diversity across the Twin Lake turbidite contact are consistent with previous studies showing that relict DNA signatures attributable to sediment transport may be more easily preserved in low‐energy, anoxic environments. This work raises the possibility that deep subsurface microbial communities may inherit variations in microbial diversity from sediment flow and deformation events.     

Transport Speed of the Mucous Feeding Filter in Clavelina lepadiformis (Aplousobranchiata,Tunicata)

The transport speed of latex spheres trapped in the mucous feeding filter of 30 specimens of Calvelina lepadiformis (Müller 1776) was measured by microscopical examination and a stop watch. The speed across the gill screen increased from 24 μm s^?1 in small animals to 220 μm s^?1 in large ones. The net translocation speed of the food roll behind the dorsal Lamina was generally much slower but revealed a similar trend (7–78 μm s^?1). The increased feeding filter speed in larger animals was parallelled by an increased branchial circumference in such a way that the duration of water filtration through each part of the filter remained almost constant (between 38 and 56 s as means for all groups of animals). The amount of filter secreted by large animals approached 150 cm² h^?1 or 2.5 m² week^?1. Based on available data on water filtration rates it could be calculated that the speed of water through the filter was of the same order of magnitude as the observed translocation speed of the filter.