Golgi apparatus isolation and use in cell-free systems

Summary Recent advances in understanding the molecular mechanisms of membrane traffic to and through the Golgi apparatus have been predicated in large measure on the use of permeabilized animal cells, and on completely cell-free systems. These systems have included those addressing inter-Golgi apparatus membrane traffic, endoplasmic reticulum to Golgi apparatus traffic, and endocytotic events. Development of cell-free systems depends on the use of isolated fractions. Specificity is often achieved by using a compartment-specific assay so that the fractions employed can be very crude. More recently cell-free systems also have evolved which employ highly purified and well-characterized cell fractions. The latter may be utilized in the absence of a compartment-specific assay but may require employment of compartment-specific assays for validation. Central to development of cell-free systems for membrane analysis has been the availability of isolated Golgi apparatus, first from plants and later from animal tissues and cells. A major advantage of cell-free systems is that they are most clearly amenable to the investigation of molecular mechanisms of membrane trafficking.Dedicated to Hilton H. Mollenhauer on the occasion of his retirement     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

在中国壮族家系中发现的一例-α~T/-α~Q复合β~0β~0珠蛋白基因型

本文报道在我国广西隆林壮族中发现一个罕見的HbQ复合α,β地中海贫血家系。先证者女,18岁,贫血面容,肝脾肿大。化学结构分析确证本Hb变异体为HbQ Thailand[α74(EF3)Asp→His]。血红蛋白组成以及α和β珠蛋白基因分析结果表明,先证者的珠蛋白基因型为-α~Q/-α~T复合β°/β°(IVSI-1G→T/Codon17A→T);先证者父的基因型为-‘α~Q/-复合β~O/β~A(IVSI-1G→T/β~A);先证母的基因型为-α~T/αα复合β~O/β~A(Codon17A→T/β~A)。     

Leaf nutrients in relation to stature and life form in tropical rain forest

Abstract. To document the relationship between a plant's position in the canopy and its leaf nutrient content, leaf nitrogen and phosphorus were determined for 30 species growing in mature evergreen lowland rain forest at La Selva Biological Station, Costa Rica. Species that grow either in the understory, midstory, or the canopy were selected. Species were further separated into three life forms: self-supporting monocots, self-supporting dicots, and climbers. Mass-based nutrient concentrations were expected to decrease with stature, as has been reported in studies of other forests. In fact, mass-based nitrogen and phosphorus did not vary significantly among the three adult-stature classes, although area-based values differed greatly: canopy plants averaged 60 % more nitrogen and 90 % more phosphorus per unit leaf area than understory plants. Differences in leaf characteristics were evident among the three life forms. Most notably, area-based phosphorus and leaf specific mass were lowest in climbers, intermediate in self-supporting dicots, and highest in self-supporting monocots. These results support the characterization of climbers as investing in inexpensive structures, perhaps in order to gain competitive advantage in light capture by allocating resources to maximize elongation rates.     

Nanodiamond-promoted white light emission in Eu,Dy, and Cu-containing phosphate glass: quantitative analysis and colorimetric study

A thorough analysis of glass containing Eu₂O₃ and Dy₂O₃, or Eu₂O₃, Dy₂O₃, and CuO melted together with nanodiamond powder was pursued based on measurements of optical absorption, photoluminescence (PL) emission and excitation spectra, and colorimetry. Nanodiamond facilitated the stabilization of Cu⁺ and Eu²⁺ ions with blue-emitting characteristics that, along with yellow-emitting Dy³⁺ and red-emitting Eu³⁺ led to the white light-emitting glass. Novel intensity notations implemented in intensity-based spectral ratios, and difference intensity correlation analysis were proposed for the assessment of PL properties. The chromaticity and correlated colour temperature of the emission were ultimately investigated as a two-parametric problem based on: (1) the different ionic components; and (2) the various excitation wavelengths employed. The optical analysis approach adds to the characterization methods to further fundamental understanding and provide helpful analytical tools for designing materials for tunable white light-emitting devices.     

Human urinary and blood toxicokinetics of beryllium after accidental exposure

PurposeBeryllium is known to have adverse health effects and is classified as carcinogenic to humans. However, data on systemic beryllium exposure in humans are rare and especially human toxicokinetics are largely uncharted. As such, the first reported multi-annual course of blood and urine concentrations after a high exposure scenario provides important new insights.MethodsFor a medical follow-up biomonitoring samples were collected for 56 months from a male subject after an accidental and multi-faceted high exposure. Sampling started on day 2 post-exposure for urine and day 147 for blood. The samples were analyzed by inductively coupled mass spectrometry (ICP-MS) and plotted longitudinally as a function of time. Terminal half-lives were calculated assuming a first-order elimination process.Main findingsBoth matrices showed highly increased initial concentrations (about 100-fold), despite the 147-day delay in blood sampling, and a marked decline over time. In urine, a two-phase excretion process was suspected based on the longitudinal data. Calculations gave terminal half-lives of 117.5 days and 666.5 days for phases 1 and 2, respectively. Blood kinetics called for a terminal half-life of 103.5 days. Elimination kinetics in blood and urine were comparable, simultaneously gathered samples showed an excellent correlation (R² = 0.985).Principal conclusionsThe long-term follow-up after a high initial exposure to beryllium provides the first detailed insights into the elimination course of systemically available beryllium in humans. Conform kinetics of beryllium in urine and blood and the strong correlation between both parameters indicate high data validity and support the good representation of the current systemically available beryllium by urine and blood concentration in humans. The relatively long terminal half-lives in both matrices suggest a possible accumulation in humans in case of repeated exposures.     

Protein structure dynamics by crosslinking mass spectrometry

Crosslinking mass spectrometry captures protein structures in solution. The crosslinks reveal spatial proximities as distance restraints, but do not easily reveal which of these restraints derive from the same protein conformation. This superposition can be reduced by photo-crosslinking, and adding information from protein structure models, or quantitative crosslinking reveals conformation-specific crosslinks. As a consequence, crosslinking MS has proven useful already in the context of multiple dynamic protein systems. We foresee a breakthrough in the resolution and scale of studying protein dynamics when crosslinks are used to guide deep-learning-based protein modelling. Advances in crosslinking MS, such as photoactivatable crosslinking and in-situ crosslinking, will then reveal protein conformation dynamics in the cellular context, at a pseudo-atomic resolution, and plausibly in a time-resolved manner.     

Synthesis and evaluation of the antidepressant activity of the enantiomers of bupropion

The synthesis of the enantiomers of bupropion, (rac)-2-tert-butylamino-3′-chloropropiophenone 1 (Wellbutrin®) is described. The enantiomers were compared with the racemate in both the tetrabenazine-induced sedation model and the inhibition of uptake of biogenic amine assay. No significant differences were found in their potencies to reverse tetrabenazine-induced sedation in mice or in their IC₅₀ values as inhibitors of biogenic amine uptake into nerve endings obtained from mouse brain. © 1993 Wiley-Liss, Inc.     

CryoRes: Local Resolution Estimation of Cryo-EM Density Maps by Deep Learning

Recent progress in cryo-EM research has ignited a revolution in biological macromolecule structure determination. Resolution is an essential parameter for quality assessment of a cryo-EM density map, and it is known that resolution varies in different regions of a map. Currently available methods for local resolution estimation require manual adjustment of parameters and in some cases necessitate acquisition or de novo generation of so-called “half maps”. Here, we developed CryoRes, a deep-learning algorithm to estimate local resolution directly from a single final cryo-EM density map, specifically by learning resolution-aware patterns of density map voxels through supervised training on a large dataset comprising 1,174 experimental cryo-EM density maps. CryoRes significantly outperforms all of the state-of-the-art competing resolution estimation methods, achieving an average RMSE of 2.26 Å for local resolution estimation relative to the currently most reliable FSC-based method blocres, yet requiring only the single final map as input. Further, CryoRes is able to generate a molecular mask for each map, with accuracy 12.12% higher than the masks generated by ResMap. CryoRes is ultra-fast, fully automatic, parameter-free, applicable to cryo-EM subtomogram data, and freely available at https://cryores.zhanglab.net.     

MORPHOLOGICAL EVOLUTION IN MUROID RODENTS II. CRANIOMETRIC FACTOR DIVERGENCE IN SEVEN NEOTROPICAL GENERA,WITH EXPERIMENTAL RESULTS FROM ZYGODONTOMYS

First principal components extracted from covariance matrices of log-transformed craniodental measurements closely approximate general size factors within field-collected samples representing 14 species in seven Neotropical muroid genera; because these samples are mixed-cross-sectional, scores are age-correlated and coefficients reflect postweaning growth allometries. Compared between congeners, sample first principal component coefficients are very similar, an observation that implies a nearly parallel orientation of ontogenetic trajectories in log-measurement space. On the assumption that a common general size factor (estimated as the first principal component of the pooled-within covariance matrix) accounts for most of the observed measurement covariance within samples, size-adjusted differences between congeneric species were estimated variable-by-variable in separate analyses of covariance; these differences reflect developmental adjustments of craniodental morphology that precede the measured interval of postweaning ontogeny. Vectors of size-adjusted difference coefficients are not similar from genus to genus, and a diversity of causal mechanisms is probably responsible. Analyses of captive-bred samples from two “species” of Zygodontomys provide prima facie evidence that size-adjusted differences estimated from field-collected samples have a genetic basis. Postweaning growth allometries in the muroid head skeleton may be conserved due to the biomechanical constraints of masticatory function; the apparent evolutionary plasticity of earlier ontogenetic adjustments may reflect the absence of such constraints in the fetus or suckling pup. The relevance of these results for current theories concerning the developmental genetics of mammalian morphometric evolution is discussed.