Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.     

Irreconciliation as practice: resisting impunity and closure in Argentina

In Argentina, irreconciliation is created through everyday practices of vigilance against closure and collective struggles against impunity. In this essay, I show how over several decades since the fall of the dictatorial regime (1976-83), human rights activists and laypeople have devised ways to keep the past alive while attending to injustices through embodied collective engagements with the country's history and its legacies. By examining large protests, the everyday experiences of impunity, and a filmic exploration of kinship bonds and their entanglement with civilian complicity in the repression, the essay illustrates the ways in which irreconciliation is materialized and enacted as a form of social reconstruction many years after state terrorism.     

Using multilayer network analysis to explore the temporal dynamics of collective behavior

Social organisms often show collective behaviors such as group foraging or movement.Collective behaviors can emerge from interactions between group members and may depend on the behavior of key individuals.When social interactions change over time,collective behaviors may change because these behaviors emerge from interactions among individuals.Despite the importance of,and growing interest in,the temporal dynamics of social interactions,it is not clear how to quantify changes in interactions over time or measure their stability.Furthermore,the temporal scale at which we should observe changes in social networks to detect biologically meaningful changes is not always apparent.Here we use multilayer network analysis to quantify temporal dynamics of social networks of the social spider Stegodyphus dumicola and determine how these dynamics relate to individual and group behaviors.We found that social interactions changed over time at a constant rate.Variation in both network structure and the identity of a keystone individual was not related to the mean or variance of the collective prey attack speed.Individuals that maintained a large and stable number of connections,despite changes in network structure,were the boldest individuals in the group.Therefore,social interactions and boldness are linked across time,but group collective behavior is not influenced by the stability of the social network.Our work demonstrates that dynamic social networks can be modeled in a multilayer framework.This approach may reveal biologically important temporal changes to social structure in other systems.     

Controls for Phylogeny and Robust Analysis in Pareto Task Inference

Understanding the tradeoffs faced by organisms is a major goal of evolutionary biology. One of the main approaches for identifying these tradeoffs is Pareto task inference (ParTI). Two recent papers claim that results obtained in ParTI studies are spurious due to phylogenetic dependence (Mikami T, Iwasaki W. 2021. The flipping t-ratio test: phylogenetically informed assessment of the Pareto theory for phenotypic evolution. Methods Ecol Evol. 12(4):696–706) or hypothetical p-hacking and population-structure concerns (Sun M, Zhang J. 2021. Rampant false detection of adaptive phenotypic optimization by ParTI-based Pareto front inference. Mol Biol Evol. 38(4):1653–1664). Here, we show that these claims are baseless. We present a new method to control for phylogenetic dependence, called SibSwap, and show that published ParTI inference is robust to phylogenetic dependence. We show how researchers avoided p-hacking by testing for the robustness of preprocessing choices. We also provide new methods to control for population structure and detail the experimental tests of ParTI in systems ranging from ammonites to cancer gene expression. The methods presented here may help to improve future ParTI studies.     

Dependence on prolactin of the luteolytic effect of prostaglandin F2alpha in rat luteal cell cultures

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.     

A model for proto-tRNA loading

A kinetic model for loading of proto-tRNA is presented. The kinetic parameters were first estimated from the results of Francklyn & Schimmel (1989;Nature337, 478--481), who studied the aminoacylation of both tRNA and its minihelix. Then these parameters were reduced several-fold, as is more appropriate for the prebiotic world. The simulations revealed a very slow time course of the loading reaction. We also consider a possibility for the proto-tRNA loading without a catalyst and discuss the feasibility of such processes. Analytical approximations are presented for the kinetics of proto-tRNA loading with and without enzyme. An estimate is given for the time required for the development of template- and sequence-directed systems.     

Microtubules of guard cells are light sensitive

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.     

Accumulation of heavy metals in epidermal glands of the waterlily (Nymphaeaceae)

This study investigates the anatomical aspects of heavy-metal accumulation in the waterlily (Nymphaea `Aurora', Nymphaeaceae). Epidermal glands were identified by light microscopy on the abaxial side of the leaf laminae and on the epidermis of the rhizome; glandular trichomes were observed in the petiole epidermis. Glands were not observed in the roots. Accumulation of heavy metals in these glands was monitored using a scanning electron microscope equipped for energy-dispersive spectroscopy. Further experiments showed maximal cadmium and calcium accumulation in the mature leaf lamina in daylight, and this accumulation was inhibited by the herbicide 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. These results suggest that, in Nymphaea, heavy metals are accumulated primarily in association with glands found in plant organs that have direct contact with water or mud. Deposition and storage of heavy metals by these glands may represent a stage in the sequestration and detoxification of the metals. Our results raise the possibility of utilizing waterlilies for the removal of heavy metals from polluted environments. Received: 29 April 2000 / Accepted: 8 June 2000