The role of niche measures in explaining the abundance–distribution relationship in tropical lotic chironomids

In situ measurements of both community metabolism (primary production and respiration) and PAM fluorometry were conducted during emersion on intertidal sediments in the Mont Saint-Michel Bay, in areas where oysters and mussels were cultivated. Results highlighted a low benthic metabolism compared to other intertidal areas previously investigated with the same methods. Comparisons between gross community primary production and relative electron transport rates confirmed this statement. More specifically, primary productivity remained very low all over the year, whereas the associated microalgal biomass was estimated to be high. We suggest that the microphytobenthic community studied was characterized by a self-limitation of its primary productivity by its own biomass, as previously shown in Marennes-Oléron Bay for example. The almost permanent high biomass would represent a limiting factor for micromigration processes within the first millimetres of the sediment. This could be explained by very low resuspension processes occurring in the western part of the bay, enhanced by the occurrence of numerous aquaculture structures that could decrease tidal currents in the benthic boundary layer. Handling editor: N. Desroy     

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.Cholinergic neurotransmission has key functions in life, as it regulates several central and peripheral nervous system outputs. Acetylcholine (ACh) is synthesized in the cytoplasm by the enzyme choline acetyltransferase (ChAT) (16). Choline supplied by the high-affinity choline transporter (CHT1) is required to maintain ACh synthesis (52). A lack of ChAT (4, 35) or the high-affinity choline transporter (21) in genetically modified mice is incompatible with life. ACh plays an important role in wiring the neuromuscular junction (NMJ) during development (38, 43). Embryonic synthesis of ACh is fundamental for the development of proper nerve-muscle patterning at the mammalian NMJ, as ChAT-null mice present aberrant nicotinic ACh receptor (nAChR) localization and increased motoneuron (MN) survival, axonal sprouting, and branching (4, 35).The vesicular ACh transporter (VAChT) exchanges cytoplasmic ACh for two vesicular protons (37, 41). Previously reported electrophysiological studies showed that quantal size is decreased by vesamicol, an inhibitor of VAChT, but only in nerve terminals that have been electrically stimulated (19, 59, 60, 63). VAChT overexpression in developing Xenopus MNs increases both the size and frequency of miniature-end-plate currents (54). In Caenorhabditis elegans, mutations in VAChT affect behavior (65). Moreover, a decrease in VAChT expression has functional consequences for mammals, as mutant mice with a 70% reduction in the expression levels of this transporter (VAChT knockdown [KD^HOM] mice) are myasthenic and have cognitive deficits (47). Hence, vesicular transport activity is rate limiting for neurotransmission “in vivo” (18, 47).Exocytosis of synaptic vesicle contents is the predominant mechanism for the regulated secretion of neurotransmitters (55). However, alternative mechanisms of secretion have been proposed (20, 56, 61). Quantal ACh release, comparable to that seen in developing nerve terminals, has been detected in myocytes and fibroblasts in culture, which presumably do not express VAChT (14, 24). More recently, it was found that the correct targeting of Drosophila photoreceptor axons is disrupted in flies with null mutations in ChAT (64). Remarkably, the inactivation of VAChT did not produce the same result (64). The result suggests that the release of ACh during development is not dependent on VAChT, perhaps because it is nonvesicular or because vesicular storage can occur without VAChT.To test if the VAChT-independent secretion of ACh has any physiological role in the mammalian nervous system, we generated a mouse line in which the VAChT gene is deleted. These mice lack the stimulated release of ACh from synaptosomes, die after birth, and show several alterations in neuromuscular wiring consistent with a severe decrease in the cholinergic input to muscles during development. These experiments indicate that VAChT has an important role in maintaining activity-dependent ACh release that supports life and the correct patterning of innervation at the NMJ.     

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.     

Comparative chewing efficiency in mammalian herbivores

Although the relevance of particle size reduction in herbivore digestion is widely appreciated, few studies have investigated digesta particle size across species in relation to body mass or digestive strategy. We investigated faecal particle size, which reflects the size of ingesta particles after both mastication and specialized processes such as rumination. Particle size was measured by wet sieving samples from more than 700 captive individuals representing 193 mammalian species. Using phylogenetic generalized least squares, faecal particle size scaled to body mass with an exponent of 0.22 (95% confidence interval: 0.16–0.28). In comparisons among different digestive strategies, we found that (1) equids had smaller faecal particles than other hindgut fermenters, (2) non-ruminant foregut fermenters and hindgut fermenters had similar-sized faecal particles (not significantly different), and (3) ruminants had finer faecal particles than non-ruminants. These results confirm that the relationship between chewing efficiency and body mass is modified by morphological adaptations in dental design and physiological adaptations to chewing, such as rumination. This allometric relationship should be considered when investigating the effect of body size on digestive physiology, and digestion studies should include a measure of faecal particle size.     

Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (K_MGlc = 140 μm versus K_MFrc = 1,375 μm). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H₂O₂) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H₂O₂ release. The blockage of H₂O₂ release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F₀F₁ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H₂O₂ release. Inhibition in H₂O₂ release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H₂O₂ release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H₂O₂ release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001; Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS leads to the production of superoxide radicals (O₂^·−) that can be dismutated by SOD, producing hydrogen peroxide (H₂O₂), and further decomposed by catalase and/or ascorbate-glutathione peroxidase cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress, pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).Mitochondrial ROS production is highly dependent on the membrane potential (ΔΨ_m) generated by the proton gradient formed across the inner mitochondrial membrane. High ΔΨ_m was shown to stimulate ROS production when the ETS is predominantly in a reduced state (i.e. when NADH, FADH₂, and O₂ are present in abundance but ADP or Pi levels are low). This condition is reached in resting metabolic states after a full oxidation of Glc or fatty acids. Stimulating electron flow by decreasing ΔΨ_m, either by the use of uncouplers or by coupling respiration to ATP synthesis, slows the ROS generation rate (Boveris and Chance, 1973; Korshunov et al., 1997). It has been observed that in isolated potato tuber (Solanum tuberosum) mitochondria (PTM) the uncoupling protein (referred to as PUMP in plants, or UCP in animals) causes a small decrease in ΔΨ_m when this proton carrier protein is activated by the presence of anionic fatty acids, a condition that blocks ROS generation (Vercesi et al., 1995, 2006). Nucleotides, such as ATP, antagonize this effect (Considine et al., 2003; Vercesi et al., 2006). On the other hand, fluctuations in free hexose levels due to environmental or developmental conditions (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz and Stitt, 1993) lead to variations in the oxygen consumption rate in heterotrophic tissues of plant (Brouquisse et al., 1991; Dieuaide et al., 1992). As a result, ROS-producing pathways may be either stimulated or repressed (Couée et al., 2006). Unlike PUMP activity, which is activated by an excess of free fatty acids, a specific mechanism for mitochondrial ROS production caused by an excess of hexose remains elusive.The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the hexokinase (HK):HK is a ubiquitous enzyme found in many organisms. In plants, the binding mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate that it may differ considerably from those properties described for mammal cells (Dry et al., 1983; Miernyk and Dennis, 1983; Rezende et al., 2006). It has been shown that in several mature and developing plant tissues, multiple HK isoforms are expressed with different kinetic properties and subcellular localizations. The HKs are found in cytosol, bound to the mitochondrial membrane, or in stroma of plastids in plant cells (Miernyk and Dennis, 1983; Galina et al., 1995; Damari-Weissler et al., 2007). Beyond its obvious role in glycolysis regulation, HK activity may also function as a sugar sensor, triggering a signal transduction pathway in plants (Rolland et al., 2006).In mammals, HK types I and II are associated with the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These associations were found in tissues with a high energy demand, such as heart, brain, and tumor cells (Arora and Pedersen, 1988; BeltrandelRio and Wilson, 1992; Wilson, 2003). In addition, recent evidence in mammalian cells has shown that binding of HK to VDAC located at the outer mitochondrial membrane is somehow involved in the protection against proapoptotic stimuli (Nakashima et al., 1986; Gottlob et al., 2001; Vander Heiden et al., 2001; Pastorino et al., 2002; Cesar and Wilson, 2004). Similar observations were reported for tobacco (Nicotiana tabacum) plant mitochondrial HK (mt-HK; Kim et al., 2006). However, it has been shown that drugs such as the fungicide clotrimazole and the anesthetic thiopental, which promptly disrupt the association between mt-HK and VDAC in mammalian mitochondria, are unable to promote this effect in maize (Zea mays) root mitochondria (Rezende et al., 2006). These observations suggest a different type of association of mt-HK with plant mitochondria. The binding of mt-HK with mitochondria in many plants involves a common N-terminal hydrophobic membrane anchor domain of about 24 amino acids that is related to the membrane targeting, but the exact mechanism of association is unknown (Damari-Weissler et al., 2007).Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by reducing mitochondrial ROS generation through a steady-state ADP recycling mechanism in rat brain neurons. The mitochondrial ADP recycling leads to a decrease in the ΔΨ_m coupled to the synthesis of ATP by oxidative phosphorylation (da-Silva et al., 2004; Meyer et al., 2006).Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an authentic HK activity was detected in PTM (Graham et al., 2007) and its involvement in potato tuber glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that regulates ROS formation. However, whether this association is capable of controlling the rate of ROS generation in plant mitochondria is unknown. Here, we aim to investigate the role of mt-HK activity in PTM physiology. The data indicate that mt-HK activity plays a key role as a regulator of ROS levels in respiring plant tissues exposed to high hexose levels.     

The dynamic ups and downs of genome size evolution in Brassicaceae

Crucifers (Brassicaceae, Cruciferae) are a large family comprisingsome 338 genera and c. 3,700 species. The family includes importantcrops as well as several model species in various fields ofplant research. This paper reports new genome size (GS) datafor more than 100 cruciferous species in addition to previouslypublished C-values (the DNA amount in the unreplicated gameticnuclei) to give a data set comprising 185 Brassicaceae taxa,including all but 1 of the 25 tribes currently recognized. Evolutionof GS was analyzed within a phylogenetic framework based ongene trees built from five data sets (matK, chs, adh, trnLF,and ITS). Despite the 16.2-fold variation across the family,most Brassicaceae species are characterized by very small genomeswith a mean 1C-value of 0.63 pg. The ancestral genome size (ancGS)for Brassicaceae was reconstructed as ^anc1C = 0.50 pg. Approximately50% of crucifer taxa analyzed showed a decrease in GS comparedwith the ancGS. The remaining species showed an increase inGS although this was generally moderate, with significant increasesin C-value found only in the tribes Anchonieae and Physarieae.Using statistical approaches to analyze GS, evolutionary gainsor losses in GS were seen to have accumulated disproportionatelyfaster within longer branches. However, we also found that GShas not changed substantially through time and most likely evolvespassively (i.e., a tempo that cannot be distinguished betweenneutral evolution and weak forms of selection). The data revealan apparent paradox between the narrow range of small GSs overlong evolutionary time periods despite evidence of dynamic genomicprocesses that have the potential to lead to genome obesity(e.g., transposable element amplification and polyploidy). Toresolve this, it is suggested that mechanisms to suppress amplificationand to eliminate amplified DNA must be active in Brassicaceaealthough their control and mode of operation are still poorlyunderstood.     

Physical characteristics of rumen contents in two small ruminants of different feeding type,the mouflon (Ovis ammon musimon) and the roe deer (Capreolus capreolus)

In domestic ruminants, the stratification of forestomach contents – the results of flotation and sedimentation processes – is an important prerequisite for the selective particle retention in this organ. A series of anatomical and physiological measurements suggests that the degree of this stratification varies between browsing and grazing wild ruminants. We investigated the forestomach contents of free-ranging mouflon and roe deer shot during regular hunting procedures. There was no difference between the species in the degree by which forestomach ingesta separated according to size due to buoyancy characteristics in vitro. However, forestomach fluid of roe deer was more viscous than that of mouflon, and no difference in moisture content was evident between the dorsal and the ventral rumen in roe deer, in contrast to mouflon. Hence, the forestomach milieu in roe deer appears less favourable for gas or particle separation due to buoyancy characteristics. These findings are in accord with notable differences in forestomach papillation between the two species. In roe deer, particle separation is most likely restricted to the reticulum, whereas in mouflon, the whole rumen may pre-sort particles to a higher degree. The results suggest that differences in forestomach physiology may occur across ruminant species.     

An N-terminal diacidic motif is required for the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane

Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4–6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.     

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A^–/– PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A^–/– human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo–electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A^–/– cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1^–/– cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A^–/– cilia. However, RSPH4A^–/– cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A^–/– PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.