Spatial metabolomics using imaging mass spectrometry to identify the localization of asparaptine A in Asparagus officinalis

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis.     

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.     

Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl

Cochliobolus heterostrophus Tub1 described here is the first beta-tubulin gene characterized from a naturally occurring benomyl-resistant ascomycete plant pathogen. The gene encodes a protein of 447 amino acids. The coding region of Tub1 is interrupted by three introns, of 116, 55, and 56 nt, situated after codons 4, 12, and 53, respectively. As a result of the preference for pyrimidines in the third position of the codons when a choice exists between purines and pyrimidines, codon usage in the Tub1 gene is biased. Tub1 shows high homology with beta-tubulin genes of other ascomycete species. However, Tub1 is exceptional in having Tyr(167), compared with Phe(167), possessed by beta-tubulin genes of other ascomycetes sequenced thus far. The Tyr(167) residue has been associated with benomyl resistance in other organisms. In contrast, all other benomyl-implicated residues of Tub1 correspond to sensitivity. Based on these results, we suggest that benomyl resistance in the fungus probably is attributed to Tyr(167).     

An angiotensin II AT1 receptor antagonist, telmisartan augments glucose uptake and GLUT4 protein expression in 3T3-L1 adipocytes

Evidence has accumulated that some of the angiotensin II AT1 receptor antagonists have insulin-sensitizing property. We thus examined the effect of telmisartan on insulin action using 3T3-L1 adipocytes. With standard differentiation inducers, a higher dose of telmisartan effectively facilitated differentiation of 3T3-L1 preadipocytes. Treatment of both differentiating adipocytes and fully differentiated adipocytes with telmisartan caused a dose-dependent increase in mRNA levels for PPARgamma target genes such as aP2 and adiponectin. By contrast, telmisartan attenuated 11beta-hydroxysteroid dehydrogenase type 1 mRNA level in differentiated adipocytes. Of note, we demonstrated for the first time that telmisartan augmented GLUT4 protein expression and 2-deoxy glucose uptake both in basal and insulin-stimulated state of adipocytes, which may contribute, at least partly, to its insulin-sensitizing ability.     

Possible involvement of the alpha1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.     

Computer simulations of seasonal outbreak and diurnal vertical migration of cyanobacteria

Algal blooms caused by cyanobacteria are characterized by two features with different time scales: one is seasonal outbreak and collapse of a bloom and the other is diurnal vertical migration. Our two-component mathematical model can simulate both phenomena, in which the state variables are nutrients and cyanobacteria. The model is a set of one-dimensional reaction-advection-diffusion equations, and temporal changes of these two variables are regulated by the following five factors: (1) annual variation of light intensity, (2) diurnal variation of light intensity, (3) annual variation of water temperature, (4) thermal stratification within a water column and (5) the buoyancy regulation mechanism. The seasonal change of cyanobacteria biomass is mainly controlled by factors, (1), (3) and (4), among which annual variations of light intensity and water temperature directly affect the maximum growth rate of cyanobacteria. The latter also contributes to formation of the thermocline during the summer season. Thermal stratification causes a reduction in vertical diffusion and largely prevents mixing of both nutrients and cyanobacteria between the epilimnion and the hypolimnion. Meanwhile, the other two factors, (2) and (5), play a significant role in diurnal vertical migration of cyanobacteria. A key mechanism of vertical migration is buoyancy regulation due to gas-vesicle synthesis and ballast formation, by which a quick reversal between floating and sinking becomes possible within a water column. The mechanism of bloom formation controlled by these five factors is integrated into the one-dimensional model consisting of two reaction-advection-diffusion equations.     

The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal–dependent manner.     

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis

Background

Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles.

Results

Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies.

Conclusion

Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.