Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess

The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate. Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)). No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K. lactis and other known Crabtree-negative yeasts. During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions. When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change. Our results suggest that the low tendency for ethanol formation in K. marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.     

Photosynthesis and respiration in Alocasia macrorrhiza following transfers to high and low light

Summary Photosynthetic capacities and respiration rates of Alocasia macrorrhiza leaves were measured for 4 weeks following reciprocal transfers between high (20% of full sun) and low (1% of full sun) light environments. Photosynthetic capacities and respiration rates of mature, high-light leaves were 1.7 and 4.5 times those of low-light leaves, respectively. Following transfer, respiration rates adjusted within 1 week to those characteristic of plants grown in the new environment. By contrast, photosynthetic capacities either did not adjust or changed only slowly following transfer. Most of the difference in respiration between high- and low-light leaves was related to the carbohydrate status as determined by the daily PFD and little was directly related to the maintenance costs of the photosynthetic apparatus. Leaf construction cost was directly proportional to maximum photosynthetic capacity. Consequently, although daily carbon gain per unit leaf area was the same for low-light and high to low-light transferred plants within a week after transfer, the carbon return per unit of carbon investment in the leaves remained lower in the high to low transfer plants throughout the 4 week measurement period. Conversely, in high-light, the low leaf construction cost of the low to high-light transferred plants resulted in carbon gain per unit investment just as high as that of the high-light plants.     

Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

Lactobacillus plantarum is frequently isolated from the fermentation of plant material where tannins are abundant. L. plantarum strains possess tannase activity to degrade plant tannins. An L. plantarum tannase (TanB_Lp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanA_Lp). While all 29 L. plantarum strains analyzed in the study possess the tanB_Lp gene, the gene tanA_Lp was present in only four strains. Upon methyl gallate exposure, the expression of tanB_Lp was induced, whereas tanA_Lp expression was not affected. TanA_Lp showed only 27% sequence identity to TanB_Lp, but the residues involved in tannase activity are conserved. Optimum activity for TanA_Lp was observed at 30°C and pH 6 in the presence of Ca²⁺ ions. TanA_Lp was able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanA_Lp was able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanA_Lp tannase in some L. plantarum strains provides them an advantage for the initial degradation of complex tannins present in plant environments.     

New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.     

NADPH oxidases 1 and 4 mediate cellular senescence induced by resveratrol in human endothelial cells

Resveratrol is believed to be partially responsible for the French paradox—the low risk of cardiovascular disease despite a high-fat diet in the French population. Recently, resveratrol has also been discussed as a life-span booster in several organisms. Age-related diseases are associated on the cellular level with senescence. We, therefore, hypothesized that resveratrol is vasoprotective by counteracting endothelial cell senescence. Surprisingly, we observed that chronic treatment with resveratrol (10 μM) was prosenescent in primary human endothelial cells. Resveratrol induced elevated reactive oxygen species (ROS) levels that were associated with and causally linked to an accumulation of cells in the S phase of the cell cycle, as measured by flow cytometry. We further show that cell accumulation in S phase leads to increased ROS and finally senescence. Using an siRNA approach, we clearly identified two NADPH oxidases, Nox1 and Nox4, as major targets of resveratrol and primary sources of ROS that act upstream of the observed S-phase accumulation.     

Conserved motifs in mechanosensitive channels MscL and MscS

Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Correction to: The floral biology and the role of staminal connective appendages during pollination of the endoparasite Bdallophytum americanum (Cytinaceae)

Bdallophytum americanum (Cytinaceae) is an endoparasitic plant species, meaning only the flowers emerge from the host during the reproductive season. Reports on the pollination biology of this species state that its primary pollinators are carrion flies attracted by the smell of the flowers and nectar as a reward. However, the functional role of one of the most outstanding attributes of B. americanum has been neglected. These are the staminal appendages formed by the apical overgrowth of connective tissue during anther development. To determine whether these staminal appendages play a role in pollination, we monitored a nectarless population of B. americanum. We described the inflorescence emergence, floral movements, and pollination and performed field experiments to test whether the absence of the staminal connective appendages affected the visitation frequency. Male inflorescences emerge early, and both male and female flowers open during the day and do not close. Hoverflies are the most frequent visitors to both floral sexes and carry the most pollen. Moreover, the movement of staminal appendages matching the pollen viability changes is reported for the first time. The staminal appendages are the structures where pollinators land before foraging. The field experiments showed that the visitation frequency decreased sharply without staminal appendages. As a landing platform, the staminal connective appendages in B. americanum are crucial for pollinator positioning and collecting viable pollen.