CBP70, a glycosylated nuclear lectin

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by β-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-I agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis [Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal βGal residues, Galβ1–3 GalNAc, Man α1–3 Man, sialic acid α2–6 linked to Gal or GalNAc; and sialic acid α2–3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and α-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting. J. Cell. Biochem. 66:370–385, 1997. © 1997 Wiley-Liss, Inc.     

In utero and early-life exposure of rats to a Wi-Fi signal: screening of immune markers in sera and gestational outcome

An experimental approach was used to assess immunological biomarkers in the sera of young rats exposed in utero and postnatal to non-ionizing radiofrequency fields. Pregnant rats were exposed free-running, 2 h/day and 5 days/week to a 2.45 GHz Wi-Fi signal in a reverberation chamber at whole-body specific absorption rates (SAR) of 0, 0.08, 0.4, and 4 W/kg (with 10, 10, 12, and 9 rats, respectively), while cage control rats were kept in the animal facility (11 rats). Dams were exposed from days 6 to 21 of gestation and then three newborns per litter were further exposed from birth to day 35 postnatal. On day 35 after birth, all pups were sacrificed and sera collected. The screening of sera for antibodies directed against 15 different antigens related to damage and/or pathological markers was conducted using enzyme-linked immunosorbent assay (ELISA). No change in humoral response of young pups was observed, regardless of the types of biomarker and SAR levels. This study also provided some data on gestational outcome following in utero exposure to Wi-Fi signals. Mass evaluation of dams and pups and the number of pups per litter was monitored, and the genital tracts of young rats were observed for abnormalities by measuring anogenital distance. Under these experimental conditions, our observations suggest a lack of adverse effects of Wi-Fi exposure on delivery and general condition of the animals.     

Simulation of direct shear tests on rooted and non-rooted soil using finite element analysis

The finite element (FE) method has been used in recent years to simulate overturning processes in trees and to better comprehend plant anchorage mechanics. We aimed at understanding the fundamental mechanisms of root-soil reinforcement by simulating direct shear of rooted and non-rooted soil. Two- (2D) and three-dimensional (3D) FE simulations of direct shear box tests were carried out using readily available software for routine strength assessment of the root-soil composite. Both rooted and non-rooted blocks of soil were modelled using a simplified model of root distribution and root material properties representative of real roots. Linear elastic behaviour was assumed for roots and the soil was modelled as an ideally plastic medium. FE analysis showed that direct shear tests were dependent on the material properties specified for both the soil and roots. 2D and 3D simulations of direct shear of non-rooted soil produced similar results and any differences between 2D and 3D simulations could be explained with regard to the spatial complexity of roots used in the root distribution model. The application of FE methods was verified through direct shear tests on soil with analogue roots and the results compared to in situ tests on rooted soil in field conditions.     

Probiotic strain <Emphasis Type="Italic">Bacillus subtilis</Emphasis> CU1 stimulates immune system of elderly during common infectious disease period: a randomized,double-blind placebo-controlled study

Background

Bacillus probiotics health benefits have been until now quite poorly studied in the elderly population. This study aimed to assess the effects of Bacillus subtilis CU1 consumption on immune stimulation and resistance to common infectious disease (CID) episodes in healthy free-living seniors.

Results

One hundred subjects aged 60–74 were included in this randomized, double-blind, placebo-controlled, parallel-arms study. Subjects consumed either the placebo or the probiotic (2.10⁹ B. subtilis CU1 spores daily) by short periodical courses of 10 days intermittently, alternating 18-day course of break. This scheme was repeated 4 times during the study. Symptoms of gastrointestinal and upper/lower respiratory tract infections were recorded daily by the subjects throughout the study (4 months). Blood, saliva and stool samples were collected in a predefined subset of the first forty-four subjects enrolled in the study. B. subtilis CU1 supplementation did not statistically significantly decrease the mean number of days of reported CID symptoms over the 4-month of study (probiotic group: 5.1 (7.0) d, placebo group: 6.6 (7.3) d, P?=?0.2015). However, in the subset of forty-four randomized subjects providing biological samples, we showed that consumption of B. subtilis CU1 significantly increased fecal and salivary secretory IgA concentrations compared to the placebo. A post-hoc analysis on this subset showed a decreased frequency of respiratory infections in the probiotc group compared to the placebo group.

Conclusion

Taken together, our study provides evidence that B. subtilis CU1 supplementation during the winter period may be a safe effective way to stimulate immune responses in elderly subjects.

     

Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis

Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution. E6 appears as a chameleon protein combining a conserved structural scaffold with highly variable surfaces participating in generic or specialized HPV functions. We investigated surface residues involved in two specialized activities of high-risk genital HPV E6: p53 tumor suppressor degradation and nucleic acid binding. Screening of E6 surface mutants identified an in vivo p53 degradation-defective mutant that fails to recruit p53 to ubiquitin ligase E6AP and restores high p53 levels in cervical carcinoma cells by competing with endogeneous E6. We also mapped the nucleic acid binding surface of E6, the positive potential of which correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understanding and counteracting the complex pathways of HPV-mediated pathogenesis.     

New advances on structural and biological functions of ceramide in apoptotic/necrotic cell death and cancer

Recent data on the cellular ceramide functions and its involvement in the apoptotic/necrotic cell death as well as its anticarcinogenic properties are presented. The emphasis is on the connections between the ceramide and caspase signaling pathways during the apoptotic cell death process. Notably, the experimental strategies and pharmacological tools used for establishment of the role of ceramide in triggering cell death are described. Moreover, the importance of a compartmentation of endogenous ceramide within the plasma membrane microdomains, lysosomes and mitochondria is discussed. Information on the deregulated functions of ceramide and caspase signaling pathways in several metastatic cancer types is also presented.     

A dual approach in the study of poly (ADP-ribose) polymerase: In vitro random mutagenesis and generation of deficient mice

A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory. Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity.In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting. We showed that: (i) they are exquisitely sensitive to -irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to -irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival.