Localization of trehalase in vacuoles and of trehalose in the cytosol of yeast (Saccharomyces cerevisiae)

Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.Non Standard Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - DTT dithiothreitol - MES 2-N-morpholinoethanesulfonic acid - PIPES piperazine-N,N-bis-2-ethanesulfonic acid - PMSF phenylmethylsulfonylfluoride     

Simultaneous measurement of prednisone, prednisolone and 6β-hydroxyprednisolone in urine by high-performance liquid chromatography coupled with a radioactivity detector

We describe the first method for routine measurement of prednisone, prednisolone and 6β-hydroxyprednisolone concomitantly in urine. Urine (3 ml) is extracted with ethyl acetate, washed with base and separated by high-performance liquid chromatography on a silica column with a solvent system of hexane—diethyl ether—ethanol—tetrahydrofuran—glacial acetic acid (59.9:31:2.3:6.5:0.3, v/v). The steroids are detected at 254 nm. Because no conventional internal standard was found, 6β-[³H]hydroxycortisol and [³H]prednisolone are added to urine prior to extraction; ³H is monitored by a radioactivity detector coupled with the chromatograph. The assay exhibits linearity from 200 to 7500 ng and an inter-day variability of < 11.4% (C.V.).     

Species boundaries in the messy middle—A genome‐scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.     

Synthesis of aTrichoderma chitinase which affects theSclerotium rolfsii andRhizoctonia solani cell walls

ATrichoderma sp. isolate, hereafter called T₆, produces a 46-kDa endochitinase (CHIT 46) which had been shown to drastically affectin vitro the cell walls of the phytopathogensSclerotium rolfsii andRhizoctonia solani. We attempted to gain insight into its properties. The CHIT 46 N-terminal amino acid sequence shares a very high homology with other fungal chitinases. Western blot analysis using polyclonal antibodies anti-CHIT 46 revealed that this enzyme is immunologically distinct from other proteins produced by the sameTrichoderma isolate T₆, but is immunologically identical with proteins having equivalent molar mass, probably chitinases, produced by otherTrichoderma spp. isolates. In addition, the antibodies revealed also that a substantial amount of this enzyme is secreted into the culture medium 2 d after theTrichoderma isolate T₆ comes into contact with chitin.     

Ethnobotany of the Sumu (Ulwa) of southeastern Nicaragua and comparisons with Miskitu plant lore

The Sumu (Ulwa) are one of three Amerindian groups of eastern Nicaragua. Their uses of 225 species of plants in 174 genera and 72 families were documented in two years of fieldwork. Included are 187 medicinals, 69 food plants, and 84 for other uses. Ulwa medicinals treat more than 25 human ailments, and most (80%) are native to eastern Nicaragua. Over 70% of the medicinals have a recognized bioactive principle, most are herbs (48%) or trees (33%). Leaves are the most frequently utilized plant part. Most medicinals are prepared as decoctions and are administered orally. Almost half of Ulwa food plants are domesticates, but only six are native to the New World tropics. Comparison of plant use between the Ulwa and southern Miskitu indicated that most of the species used for food (98%), medicinals (90%), and medicinal applications (80%) are the same. The Miskitu use more species, have a wider range of medicinal applications, and more unique plant uses than the Ulwa, presumably due to their larger territory. Differences in ethnobotanical usage between these groups seem to be more a reflection of scale than of remnants of cultural differences.     

Above-ground biomass references for urban trees from terrestrial laser scanning data

Background and AimsWithin extending urban areas, trees serve a multitude of functions (e.g. carbon storage, suppression of air pollution, mitigation of the ‘heat island’ effect, oxygen, shade and recreation). Many of these services are positively correlated with tree size and structure. The quantification of above-ground biomass (AGB) is of especial importance to assess its carbon storage potential. However, quantification of AGB is difficult and the allometries applied are often based on forest trees, which are subject to very different growing conditions, competition and form. In this article we highlight the potential of terrestrial laser scanning (TLS) techniques to extract highly detailed information on urban tree structure and AGB.MethodsFifty-five urban trees distributed over seven cities in Switzerland were measured using TLS and traditional forest inventory techniques before they were felled and weighed. Tree structure, volume and AGB from the TLS point clouds were extracted using quantitative structure modelling. TLS-derived AGB estimates were compared with AGB estimates based on forest tree allometries dependent on diameter at breast height only. The correlations of various tree metrics as AGB predictors were assessed.Key ResultsEstimates of AGB derived by TLS showed good performance when compared with destructively harvested references, with an R² of 0.954 (RMSE = 556 kg) compared with 0.837 (RMSE = 1159 kg) for allometrically derived AGB estimates. A correlation analysis showed that different TLS-derived wood volume estimates as well as trunk diameters and tree crown metrics show high correlation in describing total wood AGB, outperforming tree height.ConclusionsWood volume estimates based on TLS show high potential to estimate tree AGB independent of tree species, size and form. This allows us to retrieve highly accurate non-destructive AGB estimates that could be used to establish new allometric equations without the need for extensive destructive harvesting.     

Widespread use of unconventional targeting signals in mitochondrial ribosome proteins

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N‐terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS‐containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N‐terminal presequences and driven the evolution of internal targeting signals in MRPs.     

Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine–cytokine receptor binding are important to develop novel therapeutic strategies.