Expression of the alpha-thionin gene from barley in tobacco confers enhanced resistance to bacterial pathogens

Thionins are cysteine-rich, 5 kDa polypeptides which are toxic to plant pathogens in vitro. Expression of the gene encoding alpha-thionin from barley endosperm, under the 35S promoter from cauliflower mosaic virus, conferred to transgenic tobacco enhanced resistance to the bacterial plant pathogens Pseudomonas syringae pv. tabaci 153 and P. syringae pv. syringae. The barley alpha-thionin gene, which has two introns, was correctly spliced in tobacco. The alpha-thionin in transgenic plants had the expected mobility in the gradient, when separated by high- performance liquid chromatography, reacted with monospecific antibodies and showed the expected antibiotic properties in vitr

Characterization and analysis of thionin genes

The general designation of thionins has been proposed for a family of homologous proteins that have been isolated from different tissues in a wide range of plant taxa and have been variously named purothionins, viscotoxins, crambins, etc. (see Garcia-Olmedo et al., 1989). The possible involvement of thionins in plant defense was first suggested, on the basis of their in vitro toxicity to plant pathogens, by Fernandez de Caleya et al., (1972). Those observations had been prompted by earlier reports concerning the antimicrobial properties of these polypeptides (Stuart and Harris, 1942; Balls and Harris, 1944). Work on the thionins, which has been actively pursued over the past half-century, has been recently reviewed in detail (Garcia-Olmedo et al., 1989). For this reason, earlier work will only be partially summarized in the present chapter, which will focus on recent developments concerning thionin genes and their potential role in plant defense mechanisms

The thionins: a protein family that includes purothionins, viscotoxins and crambins

Almost half a century ago, a crystalline protein material was obtained from lipid extracts of wheat endosperm and designated "purothionin" (Trvpwo, wheat; 9 e i w v , sulphur) on account of its high sulphur content (Balls et al., 1942a,b). This material, which was thought to be the oxidized form of a powerful oxidation-reduction system, was found to have baetericidal and fungicidal properties (Stuart and Harris, 1942), to inhibit fermentation of wheat mashes (Balls and Harris, 1944), and to be toxic to laboratory animáis (Coulson et al., 1942)

Trypsin/α-amylase inhibitors and thionins: possible defence proteins from barley

This chapter reviews recent work on the trypsin/α-amylase inhibitor and thionin protein families. The genomic distribution of protein genes in barley and related species, gene expression and in vitro activities are considered. Some of the evidence of a possible defence role against stored products pests for inhibitors and thionins is briefly discusse

Bladder cancer index: cross-cultural adaptation into Spanish and psychometric evaluation

BACKGROUND: The Bladder Cancer Index (BCI) is so far the only instrument applicable across all bladder cancer patients, independent of tumor infiltration or treatment applied. We developed a Spanish version of the BCI, and assessed its acceptability and metric properties. METHODS: For the adaptation into Spanish we used the forward and back-translation method, expert panels, and cognitive debriefing patient interviews. For the assessment of metric properties we used data from 197 bladder cancer patients from a multi-center prospective study. The Spanish BCI and the SF-36 Health Survey were self-administered before and 12 months after treatment. Reliability was estimated by Cronbach's alpha. Construct validity was assessed through the multi-trait multi-method matrix. The magnitude of change was quantified by effect sizes to assess responsiveness. RESULTS: Reliability coefficients ranged 0.75-0.97. The validity analysis confirmed moderate associations between the BCI function and bother subscales for urinary (r = 0.61) and bowel (r = 0.53) domains; conceptual independence among all BCI domains (r ≤ 0.3); and low correlation coefficients with the SF-36 scores, ranging 0.14-0.48. Among patients reporting global improvement at follow-up, pre-post treatment changes were statistically significant for the urinary domain and urinary bother subscale, with effect sizes of 0.38 and 0.53. CONCLUSIONS: The Spanish BCI is well accepted, reliable, valid, responsive, and similar in performance compared to the original instrument. These findings support its use, both in Spanish and international studies, as a valuable and comprehensive tool for assessing quality of life across a wide range of bladder cancer patients

The CCP4 suite : integrative software for macromolecular crystallography

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world

Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation

Genitourinary tract symptoms in patients admitted with COVID-19: Exploring changes in frequency by determinants and pandemic waves

Background:: Urothelial cells exhibit increased expression of angiotensin-converting enzyme-2 receptor, which is the binding site of severe acute respiratory syndrome coronavirus 2 to cells. The frequency and distribution of genitourinary tract symptoms in patients diagnosed with coronavirus disease 2019 (COVID-19) is unknown. Objective:: We explored trends in genitourinary tract symptoms by gender and each of six pandemic waves in patients admitted for COVID-19, and related them with severity, death and length of hospitalization. Design, Setting, and Participants:: A retrospective study took place in our institution of COVID-19 admitted patients. Only patients with RT-PCR or antigen test confirmed SARS-CoV-2 infection were included. Demographic, clinical, and genitourinary symptoms were explored. Outcome Measurements and Statistical Analysis:: COVID-19 patients with genitourinary tract symptoms were compared with those without. Statistical comparisons were conducted by parametric and nonparametric tests for quantitative variables, and χ2test for qualitative variables. Results and limitations:: Out of a total of 4,661 COVID-19 patients, genitourinary symptoms were found in 21,1%. These symptoms were more frequent in patients admitted for longer than 30 days, except for urinary incontinence (UI) and erectile dysfunction (ED). Acute kidney injury (AKI) and urinary tract infections (UTI) had a higher presence in the 5th (16.7%; 12.8% respectively) and 3rd wave (13.3%; 12.6% respectively). Genitourinary symptoms were higher for those patients admitted in critical care units. Frequency of AKI, UI, UTI and acute urinary retention (AUR) were higher for patients who were finally deceased (26.2%; 3.5%; 13.6% and 3.6% respectively). Conclusions:: A high frequency of genitourinary symptoms in patients admitted for COVID-19 was observed, whose frequency and distribution varied according to pandemic waves. Specific genitourinary conditions were associated with worse outcomes and poorer prognosis

Predicted models and CCP4.

In late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP