Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3′ acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.     

Daily fluctuations of titratable acidity, content of organic acids (malate and citrate) and soluble sugars of varieties and wild relatives of Ananas comosus L. growing under natural tropical conditions

The genus Ananas has its centre of origin in northern South America. In this area, several varieties of Ananas comosus are widely cultivated, and a number of wild species are found growing under variable conditions of light intensity, soil fertility and water availability. Here we report detailed daily courses of titratable acidity, and malate, citrate and free-sugars content of several cultivated varieties of A. comosus and of A. ananassoides, a closely related species growing on granitic rock-outcrops in southern Venezuela. Day-night oscillations of both malate and citrate were detected in plants growing under full sun, but malate was by far the most important organic anion associated with CAM performance in ail populations sampled. Fructose was the dominant compound in the neutral fraction, but only sucrose showed a consistent inverse relation with the cycle of titratable acidity. The diel oscillations of free sugars measured were not always enough to account for the amount of organic anions accumulated during the night. Plants cultivated under shady conditions always showed a lower night-time increase in titratable acidity and organic acids, and also smaller oscillations in the amount of free sugars than sun exposed plants. In all populations growing under full sun, osmolality increased during the night, but it was not always possible to explain these changes on the basis of variations in molar concentrations of organic acids and sugars. Besides, no diel variations in the cations K⁺, Ca²⁺ and Mg²⁺ were detected. K⁺ was always the dominant cation (K/Ca ratios ～ 19), while Mg²⁺ was always higher than Ca²⁺ (Mg/Ca ～ 2).     

Combined effects of ethanol and manganese on cultured neurons and glia

Manganese is essential for normal development and activity of the nervous tissue. Mn²⁺ ions are involved in protein synthesis and may prevent free radical damage. Since it is now established that alcohol degradation may produce free radicals, we studied the effect of Mn²⁺ on ethanol induced alterations using cultured nerve cells as an experimental model of the central nervous system. Neurons and glial cells were cultured from rat brain cortex; a tumoral rat glial cell line (C6) was also examined. We measured enzymatic markers of nerve cell maturation (enolase, glutamine synthetase) and superoxide dismutase, a scavenger of free radicals; all these enzymes being activated by Mn²⁺ ions. Only for the glial cell types an alcohol antagonizing effect was found when Mn²⁺ was combined with ethanol. Neurons were not sensitive to that Mn²⁺ effect.     

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca²⁺-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.     

Calcitonin gene-related peptide and substance P in the pharynx and lung of the bullfrog,Rana catesbeiana

Indirect double immunofluorescence labelling in the pharynx and lung of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of two neuropeptides. In the pharynx, immunoreactive calcitonin gene-related peptide (CGRP) and substance P (SP) were localized in nerve fibers distributed within and just beneath the ciliated epithelium. In the lung, CGRP and SP were localized in nerve fibers in five principal locations: 1) within the smooth muscle layer in the interfaveolar septa; 2) in the luminal thickened edges of the septa; 3) around the pulmonary vasculature; 4) within, and 5) under the ciliated epithelium. Within the smooth muscle layer in the septa, luminal thickened septa, and around blood vessels, almost all fibers showed coexistence of CGRP and SP. Within and just beneath the ciliated epithelium in the thickened septa, all fibers showed coexistence of CGRP and SP. No immunoreactivity for vasoactive intestinal polypeptide, neuropeptide Y, galanin, somatostatin, FMRFamide, and leucine-and methionine-enkephalins was detected in the nerve fibers within the larynx and the lung. Together with our previous data, the present findings suggest that peptidergic mechanisms are involved in the regulation of amphibian respiratory systems throughout their life.     

Iron uptake in Pseudomonas aeruginosa mediated by N-(2,3-dihydroxybenzoyl)-L-serine and 2,3-dihydroxybenzoic acid

Abstract Pseudomonas aeruginosa is known to have an inducible uptake system for the enterobacterial siderophore enterobactin. In this work we have examined iron transport mediated by the biosynthetic precursor 2,3-dihydroxybenzoic acid and N -(2,3-dihydroxybenzoyl)- l -serine, a breakdown product of enterobactin. Iron complexed with 2,3-dihydroxybenzoyl-L-serine was transported into P. aeruginosa IA1 via a transport system which is energy-dependent and iron-repressible. The rate of transport was not altered by growing the cells in the presence of either pyoverdin or pyochelin, which have been shown previously to induce transport via that system. Growth of the cells in the presence of enterobactin did cause an increase in the rate of transport, indicating that the complex can be transported by the inducible enterobactin uptake system, but also that a separate system must exist. In contrast, transport of iron complexed with 2,3-dihydroxybenzoic acid was neither iron-repressible nor strongly energy-dependent, from which we conclude that there must be a novel mode of transport not characteristic of iron-siderophore transport systems.     

Effects of total flavonoids of raspberry on perimenopausal model in mice

To study the effect of raspberry total flavonoids on perimenopausal model in mice. Blank group, sham operation, and the rest of the mice made the menopausal model. Choose 72 mice castrated completely random divided into 6 groups for the experiment, respectively: model group, gengnianan (GNA) capsule group, soybean isoflavone soft (SIS) capsule group, high, mid and low dose group of total flavonoids of raspberry (TFR). Animals in each group were given the corresponding drugs tenth days after operation, and were given intragastrical administration of once a day for continuous administration of 21 days. Each group of mice in the administration of 18 days to determine the number of autonomic activities within 5 min, in the administration of 19–20 days to determine the incubation of the mice first entry into the darkroom and the number of shocks into the darkroom within 5 min. At 2 h after the last administration (fasting for 12 h), mice were sacrificed and serum was collected. Serum levels of E2, T, LH and FSH were measured. Dissect the uterus, uterus, thymus and spleen. Weigh the wet weight and calculate the organ index, the morphological changes of uterus, thymus and spleen were observed. The results showed that the TFR had a good therapeutic effect on the perimenopausal model of mice after giving a high, mid and low dose of raspberry flavonoids for some time.     

Protection from SARS-CoV-2 Delta one year after mRNA-1273 vaccination in rhesus macaques coincides with anamnestic antibody response in the lung

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Introduction: Metals in Biology: α-Ketoglutarate/Iron-Dependent Dioxygenases

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.     

Single‐molecule force spectroscopy applied to heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single‐molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single‐molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non‐risk heparin drugs or platelet‐surface and platelet‐platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.