A recombinant β-O-glucosidase from Caldocellum saccharolyticum to hydrolyse desulfo-glucosinolates

Glucosinolates, natural compounds found in Brassicaceae, can easily be transformed into desulfo-glucosinolates by action of Helix pomatia sulfatase. The recombinant -O-glucosidase from Caldocellum saccharolyticum does not catalyse glucosinolate degradation but can hydrolyse desulfo-glucosinolates (thio-d-glucosidic substrates) to produce the corresponding pure nitriles, including valuable homochiral representatives.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Exosomes: Revisiting their role as “garbage bags”

In recent years, the term “extracellular vesicle” (EV) has been used to define different types of vesicles released by various cells. It includes plasma membrane‐derived vesicles (ectosomes/microvesicles) and endosome‐derived vesicles (exosomes). Although it remains difficult to evaluate the compartment of origin of the two kinds of vesicles once released, it is critical to discriminate these vesicles because their mode of biogenesis is probably directly related to their physiologic function and/or to the physio‐pathologic state of the producing cell. The purpose of this review is to specifically consider exosome secretion and its consequences in terms of a material loss for producing cells, rather than on the effects of exosomes once they are taken up by recipient cells. I especially describe one putative basic function of exosomes, that is, to convey material out of cells for off‐site degradation by recipient cells. As illustrated by some examples, these components could be evacuated from cells for various reasons, for example, to promote “differentiation” or enhance homeostatic responses. This basic function might explain why so many diseases have made use of the exosomal pathway during pathogenesis.     

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.     

2-Amino-alpha-2'-deoxyadenosine increased duplex stability of methoxyethylphosphoramidate alpha-oligodeoxynucleotides with RNA target

A new efficient synthesis of 2-amino-alpha-2'-deoxyadenosine and its incorporation into methoxyethylphosphoramidate alpha-oligodeoxynucleotides (ODNs) via H-phosphonate chemistry were reported. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed between these analogues and their RNA target (+2 degrees C/NH2A) relative to adenosine-containing phosphoramidate alpha-oligonucleotides. Concerning the binding specificity of these modified ODNs, unlike natural ODNs, discrimination against G pairing is higher and against C pairing is lower.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Fragments and dust after Holmium laser lithotripsy with or without “Moses technology”: How are they different?

Urinary stones can be readily disintegrated by Holmium:YAG laser (Holmium laser lithotripsy), resulting in a mixture of small stone dust particles, which will spontaneously evacuate with urine and larger residual fragments (RF) requiring mechanical retrieval. Differences between fragments and dust have not been well characterized. Also, it remains unknown how the recently introduced “Moses technology” may alter stone disintegration products. Three complementary analytical techniques have been used in this study to offer an in‐depth characterization of disintegration products after in vitro Holmium laser lithotripsy: stereoscopic microscopy, scanning electron microscopy and Fourier‐transform infrared spectroscopy. Dust was separated from fragments based on its floating ability in saline irrigation. Depending on initial crystalline constituents, stone dust either conserved attributes found in larger RFs or showed changes in crystalline organization. These included conversion of calcium oxalate dihydrate towards calcium oxalate monohydrate, changes in carbapatite spectra towards an amorphous phase, changes of magnesium ammonium phosphate towards a differing amorphous and crystalline phase and the appearance of hydroxyapatite on brushite fragments. Comparatively, “Moses technology” produced more pronounced changes. These findings provide new insights suggesting a photothermal effect occurring in Holmium laser lithotripsy. Figure: Appearance of hydroxyapatite hexagons on stone dust collected after Holmium laser lithotripsy of a brushite stone using “Moses technology.”      

Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.     

Methods for the study of amphipod swimming: behavior,morphology, and fluid dynamics

A thorough hydrodynamic approach to the study of swimming in amphipods demands a multipronged attack. A possible first step would be to gather swimming behavior data and determine the biomechanics and kinematics of pleopod beat. This requires careful observation of the swimming modes, swimming speeds, body positions and other aspects of behavior and limb motion that are crucial to swimming. Secondly, it is important to describe the morphology of the body and swimming appendages. Detailed drawings of body shape and design, skeletomusculature, condylic structure, and setal density and distribution on the pleopods and pereopods, are the tools required to ascribe hydrodynamic function to specific limb and body morphology. Finally, the information gathered from behavioral observations bolstered by functional morphology studies is applied to fluid dynamic calculations of drag, lift, and thrust. The theoretical calculations are then compared with empirical determinations of drag, wake generation, vortex shedding frequency, and flow patterns around an amphipod. The fluid dynamic facet of this research is the most challenging and requires an excellent grasp of the fundamental concepts of fluid flow and access to some highly technical equipment. The proposed tripartite approach for the study of amphipod swimming is by no means an exhaustive review of all the techniques that can be employed to quantify amphipod swimming. It will nevertheless permit a rigorous and systematic study of amphipod swimming.