Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.     

Cloning of the Malic Enzyme Gene from Corynebacterium glutamicum and Role of the Enzyme in Lactate Metabolism

Malic enzyme is one of at least five enzymes, known to be present in Corynebacterium glutamicum, capable of carboxylation and decarboxylation reactions coupling glycolysis and the tricarboxylic acid cycle. To date, no information is available concerning the physiological role of the malic enzyme in this bacterium. The malE gene from C. glutamicum has been cloned and sequenced. The protein encoded by this gene has been purified to homogeneity, and the biochemical properties have been established. Biochemical characteristics indicate a decarboxylation role linked to NADPH generation. Strains of C. glutamicum in which the malE gene had been disrupted or overexpressed showed no detectable phenotype during growth on either acetate or glucose, but showed a significant modification of growth behavior during lactate metabolism. The wild type showed a characteristic brief period of exponential growth on lactate followed by a linear growth period. This growth pattern was further accentuated in a malE-disrupted strain (ΔmalE). However, the strain overexpressing malE maintained exponential growth until all lactate had been consumed. This strain accumulated significantly larger amounts of pyruvate in the medium than the other strains.     

Soil organic matter molecular composition and state of decomposition in three locations of the European Arctic

Increased mineralization of the organic matter (OM) stored in permafrost is expected to constitute the largest additional global warming potential from terrestrial ecosystems exposed to a warmer climate. Chemical composition of permafrost OM is thought to be a key factor controlling the sensitivity of decomposition to warming. Our objective was to characterise OM from permafrost soils of the European Arctic: two mineral soils—Adventdalen, Svalbard, Norway and Vorkuta, northwest Russia—and a “palsa” (ice-cored peat mound patterning in heterogeneous permafrost landscapes) soil in Neiden, northern Norway, in terms of molecular composition and state of decomposition. At all sites, the OM stored in the permafrost was at an advanced stage of decomposition, although somewhat less so in the palsa peat. By comparing permafrost and active layers, we found no consistent effect of depth or permafrost on soil organic matter (SOM) chemistry across sites. The permafrost-affected palsa peat displayed better preservation of plant material in the deeper layer, as indicated by increasing contribution of lignin carbon to total carbon with depth, associated to decreasing acid (Ac) to aldehyde (Al) ratio of the syringyl (S) and vanillyl (V) units, and increasing S/V and contribution of plant-derived sugars. By contrast, in Adventdalen, the Ac/Al ratio of lignin and the Alkyl C to O-alkyl C ratio in the NMR spectra increased with depth, which suggests less oxidized SOM in the active layer compared to the permafrost layer. In Vorkuta, SOM characteristics in the permafrost profile did not change substantially with depth, probably due to mixing of soil layers by cryoturbation. The composition and state of decomposition of SOM appeared to be site-specific, in particular bound to the prevailing organic or mineral nature of soil when attempting to predict the SOM proneness to degradation. The occurrence of processes such as palsa formation in organic soils and cryoturbation should be considered when up-scaling and predicting the responses of OM to climate change in arctic soils.     

Soil microbial diversity affects soil organic matter decomposition in a silty grassland soil

Soil microorganisms play a pivotal role in soil organic matter (SOM) turn-over and their diversity is discussed as a key to the function of soil ecosystems. However, the extent to which SOM dynamics may be linked to changes in soil microbial diversity remains largely unknown. We characterized SOM degradation along a microbial diversity gradient in a two month incubation experiment under controlled laboratory conditions. A microbial diversity gradient was created by diluting soil suspension of a silty grassland soil. Microcosms containing the same sterilized soil were re-inoculated with one of the created microbial diversities, and were amended with ¹³C labeled wheat in order to assess whether SOM decomposition is linked to soil microbial diversity or not. Structural composition of wheat was assessed by solid-state ¹³C nuclear magnetic resonance, sugar and lignin content was quantified and labeled wheat contribution was determined by ¹³C compound specific analyses. Results showed decreased wheat O-alkyl-C with increasing microbial diversity. Total non-cellulosic sugar-C derived from wheat was not significantly influenced by microbial diversity. Carbon from wheat sugars (arabinose-C and xylose-C), however, was highest when microbial diversity was low, indicating reduced wheat sugar decomposition at low microbial diversity. Xylose-C was significantly correlated with the Shannon diversity index of the bacterial community. Soil lignin-C decreased irrespective of microbial diversity. At low microbial diversity the oxidation state of vanillyl–lignin units was significantly reduced. We conclude that microbial diversity alters bulk chemical structure, the decomposition of plant litter sugars and influences the microbial oxidation of total vanillyl–lignins, thus changing SOM composition.     

Diffusion of Ofloxacin in the Endocarditis Vegetation Assessed with Synchrotron Radiation UV Fluorescence Microspectrocopy

The diffusion of antibiotics in endocarditis vegetation bacterial masses has not been described, although it may influence the efficacy of antibiotic therapy in endocarditis. The objective of this work was to assess the diffusion of ofloxacin in experimental endocarditis vegetation bacterial masses using synchrotron-radiation UV fluorescence microspectroscopy. Streptococcal endocarditis was induced in 5 rabbits. Three animals received an unique IV injection of 150 mg/kg ofloxacin, and 2 control rabbits were left untreated. Two fluorescence microscopes were coupled to a synchrotron beam for excitation at 275 nm. A spectral microscope collected fluorescence spectra between 285 and 550 nm. A second, full field microscope was used with bandpass filters at 510–560 nm. Spectra of ofloxacin-treated vegetations presented higher fluorescence between 390 and 540 nm than control. Full field imaging showed that ofloxacin increased fluorescence between 510 and 560 nm. Ofloxacin diffused into vegetation bacterial masses, although it accumulated in their immediate neighborhood. Fluorescence images additionally suggested an ofloxacin concentration gradient between the vegetation peripheral and central areas. In conclusion, ofloxacin diffuses into vegetation bacterial masses, but it accumulates in their immediate neighborhood. Synchrotron radiation UV fluorescence microscopy is a new tool for assessment of antibiotic diffusion in the endocarditis vegetation bacterial masses.     

A Biomimetic Collagen Derived Peptide Exhibits Anti-Angiogenic Activity in Triple Negative Breast Cancer

We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.     

Role of nucleotide hydrolysis in the polymerization of actin and tubulin

Here is presented a short survey of the main aspects of the involvement of nucleotide hydrolysis in the polymerization of actin and microtubules: 1) XTP hydrolysis is not tightly coupled to the polymerization process; XTP hydrolysis and phosphate release generate an unstable XDP-polymer which is maintained at steady state, in the presence of XTP, by terminal XTP-subunits; this feature can generate patterns of phase transitions of the polymer between stable and unstable conformations; 2) Interactions between subunits are involved in the mechanism of XTP hydrolysis; 3) XTP cleavage on the polymer is followed by the slow release of P_i; the structural and thermodynamic characteristics of the transient XDP-P_i-polymer may play a crucial role in the regulation of the dynamics of microtubules and actin filaments.     

Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary ¹H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu³⁷, Leu²³, Val⁴⁷, Tyr¹⁴, Trp⁴⁵ and Tyr⁵. The Trp⁴⁵ and Tyr⁵ rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986.