A Proline-Rich Motif Downstream of the Receptor Binding Domain Modulates Conformation and Fusogenicity of Murine Retroviral Envelopes

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential β-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.     

Deletions in the Parkin gene and genetic heterogeneity in a Greek family with early onset Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease and is manifested as a movement disorder. A positive family history is the second most important risk factor for developing the illness, after age. Both autosomal dominant and recessive forms of the illness have been described. Recently deletions in a novel gene, parkin, have been associated with the autosomal recessive form of the illness in Japanese families. In this study, we demonstrate that deletions of exons 5, 6 and 7 of the parkin gene are present in two affected individuals of a Greek pedigree with early onset Parkinson’s disease. However, no deletions were identified in a different branch of the same pedigree with three affected individuals. These results suggest that deletions in the parkin gene will be found in other families besides those of Japanese origin and that there must be at least one additional locus responsible for early onset autosomal recessive Parkinson’s disease. Received: 9 June 1998 / Accepted: 10 August 1998     

Temperature-sensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis

Summary Cessation of DNA synthesis in the temperature sensitive mutant 167 tsA 13 of Bacillus subtilis is correlated with the disappearance of dCTP and dATP pools at the nonpermissive temperature; dGTP and dTTP residual pools are stable. In the presence of AdR and CdR at 45°C, the dCTP and dATP pools remain normal and the cells continue to synthesise DNA and grow. It is inferred that in 167 tsA 13 AdR and CdR kinases exist, that the deoxynucleotide kinases function normally and the ribonucleotide reduction is deficient. B. subtilis strains have a hydroxyurea sensitive reductase and the drug inhibition can be reversed by exogenous deoxynucleosides. Evidence that the tsA 13 mutation is in the structural gene of the ribonucleotide reductase is discussed.     

Engineered silver nanoparticles are sensed at the plasma membrane and dramatically modify the physiology of Arabidopsis thaliana plants

Silver nanoparticles (Ag NPs) are the world's most important nanomaterial and nanotoxicant. The aim of this study was to determine the early stages of interactions between Ag NPs and plant cells, and to investigate their physiological roles. We have shown that the addition of Ag NPs to cultivation medium, at levels above 300 mg L^?1, inhibited Arabidopsis thaliana root elongation and leaf expansion. This also resulted in decreased photosynthetic efficiency and the extreme accumulation of Ag in tissues. Acute application of Ag NPs induced a transient elevation of [Ca²⁺]_cyt and the accumulation of reactive oxygen species (ROS; partially generated by NADPH oxidase). Whole‐cell patch‐clamp measurements on root cell protoplasts demonstrated that Ag NPs slightly inhibited plasma membrane K⁺ efflux and Ca²⁺ influx currents, or caused membrane breakdown; however, in excised outside‐out patches, Ag NPs activated Gd³⁺‐sensitive Ca²⁺ influx channels with unitary conductance of approximately 56 pS. Bulk particles did not modify the plasma membrane currents. Tests with electron paramagnetic resonance spectroscopy showed that Ag NPs were not able to catalyse hydroxyl radical generation, but that they directly oxidized the major plant antioxidant, l ‐ascorbic acid. Overall, the data presented shed light on mechanisms of the impact of nanosilver on plant cells, and show that these include the induction of classical stress signalling reactions (mediated by [Ca²⁺]_cyt and ROS) and a specific effect on the plasma membrane conductance and the reduced ascorbate.     

Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex

Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.     

The reactivity of heme in biological systems: autocatalytic formation of both tyrosine-heme and tryptophan-heme covalent links in a single protein architecture

We have previously shown that introduction of an engineered Met160 residue in ascorbate peroxidase (S160M variant) leads to the formation of a covalent link between Met160 and the heme vinyl group [Metcalfe, C. L., et al. (2004) J. Am. Chem. Soc. 126, 16242-16248]. In this work, we have used electronic spectroscopy, HPLC, and mass spectrometry to show that the introduction of a tyrosine residue at the same position (S160Y variant) leads, similarly, to the formation of a heme-tyrosine covalent link in an autocatalytic reaction that also leads to formation of a second covalent link from the heme to Trp41 [Pipirou, Z., et al. (2007) Biochemistry 46, 2174-2180]. Stopped-flow and EPR data implicate the involvement of a tyrosyl radical in the reaction mechanism. The results indicate that the heme can support the formation of different types of covalent links under appropriate conditions. The generality of this idea is discussed in the context of other heme enzymes.     

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.     

High-resolution, human–bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments. Received: 6 August 1998 / Accepted: 28 October 1998     

Bioinspiration as a method of problem‐based STEM education: A case study with a class structured around the COVID‐19 crisis

Bioinspiration is a promising lens for biology instruction as it allows the instructor to focus on current issues, such as the COVID‐19 pandemic. From social distancing to oxygen stress, organisms have been tackling pandemic‐related problems for millions of years. What can we learn from such diverse adaptations in our own applications? This review uses a seminar course on the COVID‐19 crisis to illustrate bioinspiration as an approach to teaching biology content. At the start of the class, students mind‐mapped the entire problem; this range of subproblems was used to structure the biology content throughout the entire class. Students came to individual classes with a brainstormed list of biological systems that could serve as inspiration for a particular problem (e.g., absorptive leaves in response to the problem of toilet paper shortages). After exploration of relevant biology content, discussion returned to the focal problem. Students dug deeper into the literature in a group project on mask design and biological systems relevant to filtration and transparency. This class structure was an engaging way for students to learn principles from ecology, evolution, behavior, and physiology. Challenges with this course design revolved around the interdisciplinary and creative nature of the structure; for instance, the knowledge of the participants was often stretched by engineering details. While the present class was focused on the COVID‐19 crisis, a course structured through a bioinspired approach can be applied to other focal problems, or subject areas, giving instructors a powerful method to deliver interdisciplinary content in an integrated and inquiry‐driven way.