Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO₂ on growth, CO₂-fixation and N₂-fixation rates by strains of the unicellular marine N₂-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO₂-acclimated cultures (190 ppm) of WH0401, growth, CO₂-fixation and N₂-fixation rates were significantly lower than those in cultures acclimated to higher (present-day ～385 ppm, or future ～750 ppm) pCO₂ treatments. Growth rates were not significantly different, however, in low-pCO₂-acclimated cultures of WH0402 in comparison with higher pCO₂ treatments. Unlike previous reports for C. watsonii (strain WH8501), N₂-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO₂. Both light and pCO₂ had a significant negative effect on gross : net N₂-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO₂. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO₂, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO₂-fixation rates of C. watsonii may be positively affected by elevated light and pCO₂ within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.     

Complex patterns of dopamine‐related gene expression in the ventral tegmental area of male zebra finches relate to dyadic interactions with long‐term female partners

Dopaminergic projections from the ventral tegmental area (VTA) to multiple efferent targets are implicated in pair bonding, yet the role of the VTA in the maintenance of long‐term pair bonds is not well characterized. Complex interactions between numerous neuromodulators modify activity in the VTA, suggesting that individual differences in patterns of gene expression in this region may explain individual differences in long‐term social interactions in bonded pairs. To test this hypothesis we used RNA‐seq to measure expression of over 8000 annotated genes in male zebra finches in established male‐female pairs. Weighted gene co‐expression network analysis identified a gene module that contained numerous dopamine‐related genes with TH found to be the most connected gene of the module. Genes in this module related to male agonistic behaviors as well as bonding‐related behaviors produced by female partners. Unsupervised learning approaches identified two groups of males that differed with respect to expression of numerous genes. Enrichment analyses showed that many dopamine‐related genes and modulators differed between these groups, including dopamine receptors, synthetic and degradative enzymes, the avian dopamine transporter and several GABA‐ and glutamate‐related genes. Many of the bonding‐related behaviors closely associated with VTA gene expression in the two male groups were produced by the male's partner, rather than the male himself. Collectively, results highlight numerous candidate genes in the VTA that can be explored in future studies and raise the possibility that the molecular/genetic organization of the VTA may be strongly shaped by a social partner and/or the strength of the pair bond.     

Analysis of purine receptor expression and functionality in alveolar epithelial cells

Despite its fundamental role in providing an extensive surface for gas exchange, the alveolar epithelium (AE) serves as an immunological barrier through, e.g., the release of proinflammatory cytokines and secretion of surfactant to prevent alveolar collapse. Thus, AE is important for sustaining lung homeostasis. Extracellular ATP secreted by alveolar epithelial cells (AECs) is involved in physiological and pathological conditions and acts mainly through the activation of purine receptors (P2Rs). When studying P2R-mediated processes, primary isolated type II AECs (piAECs) still represent the gold standard in in vitro research, although their preparation is time-consuming and requires the sacrifice of many animals. Hence, cultivated immortalized and tumor-derived AEC lines may constitute a valuable alternative. In this work, we examined P2R expression and functionality in piAECs, in immortalized and tumor-derived AEC lines with the purpose of gaining a better understanding of purinergic signaling in different cell systems and assisting researchers in the choice of a suitable cell line with a certain P2R in demand. We combined mRNA and protein analysis to evaluate the expression of P2R. For pharmacological testing, we conducted calcium ([Ca²⁺]) measurements and siRNA receptor knockdown. Interestingly, the mRNA and protein levels of P2Y₂, P2Y_6, and P2X₄ were detected on all cell lines. Concerning functionality, P2XR could be narrowed to L2 and piAECs while P2YR were active in all cell lines.

     

Soil amendment interacts with invasive grass and drought to uniquely influence aboveground versus belowground biomass in aridland restoration

Water‐holding soil amendments such as super‐absorbent polymer (SAP) may improve native species establishment in restoration but may also interact with precipitation or invasive species such as Bromus tectorum L. (cheatgrass or downy brome) to influence revegetation outcomes. We implemented an experiment at two sites in Colorado, U.S.A., in which we investigated the interactions of drought (66% reduction of ambient rainfall), B. tectorum seed addition (BRTE, 465 seeds/m²), and SAP soil amendment (25 g/m²) on initial plant establishment and 3‐year aboveground and belowground biomass and allocation. At one site, SAP resulted in higher native seeded species establishment but only with ambient precipitation. However, by the third year, we detected no SAP effects on native seeded species biomass. Treatments interacted to influence aboveground and belowground biomass and allocation differently. At one site, a SAP × precipitation interaction resulted in lower belowground biomass in plots with SAP and drought (61.7 ± 7.3 g/m²) than plots with drought alone (91.6 ± 18.1 g/m²). At the other site, a SAP × BRTE interaction resulted in higher belowground biomass in plots with SAP and BRTE (56.6 ± 11.2 g/m²) than BRTE alone (35.0 ± 3.7 g/m²). These patterns were not reflected in aboveground biomass. SAP should be used with caution in aridland restoration because initial positive effects may not translate to long‐term benefits, SAP may uniquely influence aboveground versus belowground biomass, and SAP can interact with environmental variables to impact developing plant communities in positive and negative ways.     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

ASHG 2020 Curt Stern Award introduction: Fowzan Sami Alkuraya

This article is based on the address given by the author at the 2020 virtual meeting of the American Society of Human Genetics (ASHG) on October 26, 2020. The video of the original address can be found at the ASHG website.