An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.     

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.     

Isolation and mapping of polymorphic microsatellites in cattle

Summary
A partial plasmid library with bovine genomic inserts of about 500 basepairs was screened with a (dC-dA)_n-(dG-dT)_n oligonucleotide probe for the repeated nucleotide motif (CA)_n. Eleven positive clones (0.3% of all colonies screened) were discovered and were subsequently isolated and sequenced. Eight microsatellite loci were analysed, one with eight alleles, one with seven alleles, three with six alleles, one with three alleles and two with two alleles. Six of these microsatellites were mapped by PCR-analysis of a panel of somatic hybrid lines.     

Inference in response-adaptive clinical trials when the enrolled population varies over time

A common assumption of data analysis in clinical trials is that the patient population, as well as treatment effects, do not vary during the course of the study. However, when trials enroll patients over several years, this hypothesis may be violated. Ignoring variations of the outcome distributions over time, under the control and experimental treatments, can lead to biased treatment effect estimates and poor control of false positive results. We propose and compare two procedures that account for possible variations of the outcome distributions over time, to correct treatment effect estimates, and to control type-I error rates. The first procedure models trends of patient outcomes with splines. The second leverages conditional inference principles, which have been introduced to analyze randomized trials when patient prognostic profiles are unbalanced across arms. These two procedures are applicable in response-adaptive clinical trials. We illustrate the consequences of trends in the outcome distributions in response-adaptive designs and in platform trials, and investigate the proposed methods in the analysis of a glioblastoma study.     

Balancing photosynthetic light-harvesting and light-utilization capacities in potato leaf tissue during acclimation to different growth temperatures

We investigated the effect of temperature during growth and development on the relationship between light-harvesting capacity, indicated by chlorophyll concentration, and light-utilization potential, indicated by light- and bicarbonate-saturated photosynthetic oxygen evolution, in Solanum tuberosum L. cv. Norland. Conal plantles were transplanted and grown at 20°C for 2 weeks before transfer to 12, 16, 20, 24 and 28°C for 6 weeks. After 4 weeks of the temperature treatments, leaf tissue fresh weights per area were one-third higher in plants grown at 12°C vs those grown at 28°C. Conversely, chlorophyll content per area in tissue grown at 12°C was less than one-half of that of tissue grown at 28°C at 4 weeks. Photosynthetic capacity measured at a common temperature of 20°C and expressed on a chlorophyll basis was inversely proportional to growth temperature. Leaf tissue from plants grown at 12°C for 4 weeks had photosynthetic rates that were 3-fold higher on a chlorophyll basis than comparable tissue from plants grown at 28°C. These results suggest that the relationship between light-harvesting capacity and light-utilization potential varies 3-fold in response to the growth temperatures examined. The role of this response in avoidance of photoinhibition is discussed.     

In transformed tobacco cells the apoplasmic invertase inhibitor operates as a regulatory switch of cell wall invertase

Agrobacterium tumefaciens-transformed tobacco suspension-cultured cells (TSCC) exhibit no significant quantitative changes of cell wall invertase protein (CWI) during a culture period of 40 days, whereas CWI activity decreases strongly between 10 and 30 days after cell transfer to fresh medium. Western blot analysis revealed that the apoplasmic invertase inhibitor (INH) is equally expressed throughout the entire culture period. When apoplasmic protein fractions from 4 and 28 days old cell cultures are chromatographed on Concanavalin A(ConA)-Sepharose, the non-glycosylated INH always coelutes with the ConA-bound fraction, suggesting that (i) INH and the glycosylated CWI form a complex in the apoplasmic space, and (ii) INH binding is not sufficient for CWI inhibition. The high specificity of INH binding to CWI was confirmed by native cathodic polyacrylamide gel electrophoresis. Expression analysis of CWI and INH indicates that, at least during certain stages of plant development (seedlings, roots of adult plants), CWI activity may be modulated by INH, the latter operating as a regulatory switch.     

Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C²-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.     

The Origin of Trypsin: Evidence for Multiple Gene Duplications in Trypsins

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed. Received: 22 November 1997 / Accepted: 26 January 1998