Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

Characterization of gamma-aminobutyric acid-benzodiazepine receptor complexes by protection against inactivation by group-specific reagents

Abstract: The chemical topography of the γ-aminobutyric acid (GABA) and benzodiazepine (BZ) receptors was investigated in a thoroughly washed cortical membrane preparation of the rat. Chemical modification by several amino- and tyrosyl-selective reagents and the protection from it by direct and allosteric ligands of the GABA-BZ receptor complex were used to identify the residues at the binding sites. Inhibition of specific GABA binding by p -diazobenzenesulfonic acid (DSA), tetrani-tromethane (TNM), and N -acetylimidazole and the selective and complete protection from it by GABA and muscimol suggest the presence of a tyrosine residue at the GABA_A site. TNM, like DSA, selectively decreased the number of the low-affinity GABA receptors, and this could be completely protected only by GABA concentrations that can saturate the low-affinity sites. TNM pre-treatment also abolished the muscimol enhancement of [³H]diazepam binding, which suggests that the low-affinity GABA receptor sites are responsible for this enhancement. Inhibition of GABA binding by pyridoxal-5-phosphate (PLP) and the selective protection by GABA and muscimol support the presence of a lysine residue at the GABA_A receptor site. Complete and selective protection from diethylpyrocarbonate (DEP) inhibition of [³H]diazepam binding by flurazepam suggests the presence of a histidine residue at the BZ site. Flurazepam selectively protected from inhibition of [³H]diazepam binding by N -bromosuccinimide and N -acetylimidazole, but not that by DSA and TNM, which does not allow a unanimous conclusion regarding the presence of tyrosine or tryptophan residues at the BZ site.     

Catechol modification and covalent immobilization of catalase on titania submicrospheres

In this article, chemical modification with catecholic derivative in solution and subsequent immobilization of catalase (CAT) on titania submicrospheres (450–500 nm) were described. Catalase was first reacted with 3-(3,4-dihydroxyphenyl) propionic acid activated via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry. The above chemically modified CAT bearing catechol groups was then covalently bound to the surface of titania through the facile chelation reaction between the catechol groups and titania. The immobilized CAT retained 60% catalytic activity with a high loading capacity of 500 mg/g titania. Meanwhile, the immobilized CAT displayed enhanced operational stability, thermal stability and storage stability compared with native, modified CAT counterparts. In repeated batches of decomposition of hydrogen peroxide, after 10 and 19 cycles, the immobilized CAT maintained about 90% and 75% of its initial activity, respectively.     

Evidence that core histone H3 is targeted to the mitochondria in Brassica oleracea

The core histone proteins H2A, H2B, H3 (histone H3) and H4 are known to form nucleosomes with nuclear DNA, but are historically considered to be absent from mitochondria. We suggest that H3 is a dual‐targeted protein, found in mitochondria as well as N (nuclei). WoLF PSORT and MitoProt analyses of H3 sequences revealed mitochondrial targeting signals, and immunohistochemistry indicated mitochondrial distribution. Western blots of Brassica oleracea cv. Botrytis (cauliflower) mitochondrial extracts were positive for H3, when the primary antibody was against the conserved C‐terminus. MS/MS (tandem mass spectrometry) analyses confirmed the Western blot data. Interestingly, Western blots of the same mitochondrial extracts were almost completely negative for H3 when the primary antibodies were highly specific for the N‐terminal tail region of H3, suggesting that these antibodies are blocked by a modification of the tail of the H3 that occurs predominantly in the mitochondria, but not in the nucleus. Modifications of the tail of core H3 are known to help control nuclear genes. Future studies of the possible functions of mitochondrial H3 could lead to a greater understanding of the ability of a cell to synchronize nuclear and mitochondrial gene expression.     

Identification and characterization of N6-methyladenosine modification of circRNAs in glioblastoma

This research systematically profiled the global N6-methyladenosine modification pattern of circular RNAs (circRNAs) in glioblastoma (GBM). Based on RNA methylation sequencing (MeRIP sequencing or N6-methyladenosine sequencing) and RNA sequencing, we described the N6-methyladenosine modification status and gene expression of circRNAs in GBM and normal brain tissues. N6-methyladenosine–related circRNAs were immunoprecipitated and validated by real-time quantitative PCR. Bioinformatics analysis and related screening were carried out. Compared with those of the NC group, the circRNAs from GBM exhibited 1370 new N6-methyladenosine peaks and 1322 missing N6-methyladenosine peaks. Among the loci associated with altered N6-methyladenosine peaks, 1298 were up-regulated and 1905 were down-regulated. The N6-methyladenosine level tended to be positively correlated with circRNA expression. Bioinformatics analysis was used to predict the biological function of N6-methyladenosine–modified circRNAs and the corresponding signalling pathways. In addition, through PCR validation combined with clinical data mining, we identified five molecules of interest (BUB1, C1S, DTHD1, F13A1 and NDC80) that could be initial candidates for further study of the function and mechanism of N6-methyladenosine–mediated GBM development. In conclusion, our findings demonstrated the N6-methyladenosine modification pattern of circRNAs in human GBM, revealing the possible roles of N6-methyladenosine–mediated novel noncoding RNAs in the origin and progression of GBM.     

Promoting biological control in a rapidly changing world

Sustainable agriculture must provide for growing human demands for crops while minimizing impacts on ecosystems. This is a daunting challenge as agroecosystems have trended towards monocultures with intensive synthetic inputs. Moreover, agricultural landscapes often lack natural habitats that are necessary to support biodiversity. Furthermore, problems associated with agricultural intensification and land-use change may be exacerbated by climate change, which increases the frequency of disturbances, modifies the suitability of habitats, and changes the way species interact. To meet this challenge, farmers must increasingly rely on integrated pest management strategies, including biological control. Biological control of arthropods, weeds, and diseases can promote the stability and diversity of agricultural communities and aid in reducing synthetic inputs. Promoting biological control may thus help farming systems adapt to a rapidly changing world. This special issue considers how multiple global change drivers such as agricultural intensification, land-use change, and climate change affect biological control. Here, we discuss these papers and highlight concepts that remain relatively unexplored in the context of global change and biological control. Future research addressing these issues will promote biological control and enhance agricultural sustainability in a rapidly changing world.     

N-glycosylation in Archaea—New roles for an ancient posttranslational modification

Genome analysis points to N-glycosylation as being an almost universal posttranslational modification in Archaea. Although such predictions have been confirmed in only a limited number of species, such studies are making it increasingly clear that the N-linked glycans which decorate archaeal glycoproteins present diversity in terms of both glycan composition and architecture far beyond what is seen in the other two domains of life. In addition to continuing to decipher pathways of N-glycosylation, recent efforts have revealed how Archaea exploit this variability in novel roles. As well as encouraging glycoprotein synthesis, folding and assembly into properly functioning higher ordered complexes, N-glycosylation also provides Archaea with a strategy to cope with changing environments. Archaea can, moreover, exploit the apparent species-specific nature of N-glycosylation for selectivity in mating, and hence, to maintain species boundaries, and in other events where cell-selective interactions are required. At the same time, addressing components of N-glycosylation pathways across archaeal phylogeny offers support for the concept of an archaeal origin for eukaryotes. In this MicroReview, these and other recent discoveries related to N-glycosylation in Archaea are considered.     

CIGAR‐seq,a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m⁶A and m⁵C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high‐throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m⁵C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non‐overlapping subsets of mRNA m⁵C sites and together contributed to almost all the m⁵C modification in mRNA. Finally, using m¹A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification.