Immunocytochemical characterisation of endophytic bacteriaAzospirillum brasilense, Herbaspirillum seropedicae, Burkholderia ambifaria andGluconacetobacter diazotrophicus

In the present work an immunocytochemical characterisation of four endophytic bacterial species has been made by using polyclonal antiserum produced against each of the four bacterial strains previously heated at 60 °C. The aim of this researchsito identify common elements among bacteria associated with their endophytic behaviour. Analysis of extracts of each strain by immunoblotting and ELISA confirmed the presence of proteins from different bacterial strains made up of common epitopes. However, antisaproduced againstHerbaspirillum seropedicae andBurkholderia ambifaria show a high number of bands recognised on each extracts, while antisera againstAzospirillum brasilense andGluconacetobacter diazotrophicus show a low number of bands recognised on each extract. Immunogold labelling showed that epitopes are located both on the cell wall and in the cytoplasm; most likely they could be preursor cell wall proteins synthesized inside the cytoplasm and subsequently transported onto cell wall. Finally, the common bands amog bacterial strains revealed by immunoblotting could play a role as active hydrolases involved in host tissue penetration.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

Changes in ovarian follicles and in vitro sex hormone release in the lizard Podarcis sicula sicula

An in vitro superfusion method was used to test sex hormone release from different kinds of ovarian follicle (growing follicles, postovulatory follicles, and atretic follicles) in the lizard Podarcis sicula sicula. Sex hormone output changes with the stage of follicle evolution and sexual cycle. Previtellogenetic follicles prevail in early-spring quiescent ovaries and secrete mainly progesterone, which is probably utilized at that phase to delay ovarian resumption. In the active ovary, progesterone output from previtellogenetic follicles decreases, whereas vitellogenetic follicles produce a significant amount of 17β-estradiol, which is necessary for sustaining vitellogenin synthesis by the liver and oviduct growth. As follicles become ripe, progesterone production is resumed, and it increases in young postovulatory follicles. This is in line with the functions assigned to the hormone at that phase of the sexual cycle, i.e., the induction of oocyte maturation and the regulation of egg retention in the oviduct. Postovulatory follicles can also synthetize 17β-estradiol. After oviposition, this hormone, which is secreted by the old postovulatory follicles, can reinitiate vitellogenin synthesis, allowing the development of a new oocyte set. Our data confirm that active, although ephemeral, corpora lutea are also formed in oviparous species. A limited contribution to ovarian sex steroid production derives also from atretic follicles, at least at the early stages of the breeding cycle. © 1993 Wiley-Liss, Inc.     

Nalidixic acid-resistant V79 cells with reduced DNA topoisomerase II activity and amplification prone phenotype.

Spontaneously nalidixic acid-resistant lines (NAr lines) were selected from a V79 Chinese hamster cell line and phenotypically characterized. NAr lines showed an increased doubling time, a higher number of spontaneous SCE, and more interestingly, decreased DNA topoisomerase II activity. These lines were also cross-resistant to the eukaryotic topoisomerase II inhibitors etoposide and adriamycin, but showed the same level of sensitivity as the parental line to the DNA topoisomerase I inhibitor camptothecin. NAr lines were cross-resistant to other drugs, such as PALA, MTX and MPA, resistance to which has been shown to arise by amplification of the target genes. This last feature, together with enhanced cross-resistance to PALA and MTX when employed simultaneously, suggests that NAr lines have an 'amplification prone' phenotype. From these results the decreased activity of topoisomerase II seems to be involved in the generation of amplified sequences possibly by affecting recombinational events underlying gene amplification.     

Mycophenolate mofetil versus azathioprine in kidney transplant recipients on steroid-free,low-dose cyclosporine immunosuppression (ATHENA): A pragmatic randomized trial

BackgroundWe compared protection of mycophenolate mofetil (MMF) and azathioprine (AZA) against acute cellular rejection (ACR) and chronic allograft nephropathy (CAN) in kidney transplant recipients on steroid-free, low-dose cyclosporine (CsA) microemulsion maintenance immunosuppression.Methods and findingsATHENA, a pragmatic, prospective, multicenter trial conducted by 6 Italian transplant centers, compared the outcomes of 233 consenting recipients of a first deceased donor kidney transplant induced with low-dose thymoglobulin and basiliximab and randomized to MMF (750 mg twice/day, n = 119) or AZA (75 to 125 mg/day, n = 114) added-on maintenance low-dose CsA microemulsion and 1-week steroid. In patients without acute clinical or subclinical rejections, CsA dose was progressively halved. Primary endpoint was biopsy-proven CAN. Analysis was by intention to treat.Participants were included between June 2007 and July 2012 and followed up to August 2016. Between-group donor and recipient characteristics, donor/recipient mismatches, and follow-up CsA blood levels were similar. During a median (interquartile range (IQR)) follow-up of 47.7 (44.2 to 48.9) months, 29 of 87 biopsied patients on MMF (33.3%) versus 31 of 88 on AZA (35.2%) developed CAN (hazard ratio (HR) [95% confidence interval (CI)]: 1.147 (0.691 to 1.904, p = 0.595). Twenty and 21 patients on MMF versus 34 and 14 on AZA had clinical [HR (95% CI): 0.58 (0.34 to 1.02); p = 0.057) or biopsy-proven subclinical [HR (95% CI): 1.49 (0.76 to 2.92); p = 0.249] ACR, respectively. Combined events [HR (95% CI): 0.85 (0.56 to 1.29); p = 0.438], patient and graft survival, delayed graft function (DGF), 3-year glomerular filtration rate (GFR) [53.8 (40.6;65.7) versus 49.8 (36.8;62.5) mL/min/1.73 m², p = 0.50], and adverse events (AEs) were not significantly different between groups.Chronicity scores other than CAN predict long-term graft outcome. Study limitations include small sample size and unblinded design.ConclusionsIn this study, we found that in deceased donor kidney transplant recipients on low-dose CsA and no steroids, MMF had no significant benefits over AZA. This finding suggests that AZA, due to its lower costs, could safely replace MMF in combination with minimized immunosuppression.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00494741","term_id":"NCT00494741"}}NCT00494741; EUDRACT 2006-005604-14.

Piero Ruggenenti and co-workers study maintenance immunosuppression in deceased-donor kidney transplantation.