Expression and characterization of biologically active human Fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 10⁶ cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays. S. Zappitelli and L. D’Alatri contributed equally to this work.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Differential location and structural specificities of sialic acid-beta-D-Gal sequences belonging to sialoderivatives of rabbit oviduct under hormonal treatment

Sialoderivatives expressed in the rabbit oviduct under hormonal treatment have been investigated in situ by lectin histochemistry with a view to specifying further regional and temporal specializations which enable ampulla and isthmus to play distinct roles in the reproductive events. Application of MAL II and SNA lectins allowed sialoglycoconjugates containing Sia(alpha2,3)Gal and Sia(alpha2,6)Gal groups to be discriminated. Sialic acid residues linked to Gal(beta1,3)-D-GalNAc sequences were identified using PNA combined with sialidase digestion. Information on structural features of sialic acids were acquired by deacetylation and differential oxidation pretreatments. In both oviductal portions, Sia(alpha2,6) groups were restricted to the luminal surface of the lining epithelium while Sia(alpha2,3) groups were specifically located in the secretory, non-ciliated cells. In the ampullary epithelium, non-acetylated sialic acids alpha2,3-linked to Gal(beta1,3)-D-GalNAc sequences were largely present. Only at ovulation time were sialic acid residues containing acetyl substituents on C4 also found. A great variety of sialic acids were found in the isthmic epithelium which showed the highest expression of acetylated forms at the first hours after the hormonal treatment. The heterogeneity of sialoderivatives differently expressed in the ampulla and isthmus as well as their distinct cycle-dependent modulation suggest that sialylated components may contribute to the molecular and functional specificities within the oviductal epithelium.     

Monitoring for human cytomegalovirus infection in solid organ transplant recipients through antigenemia and glycoprotein N (gN) variants: evidence of correlation and potential prognostic value of gN genotypes

Human cytomegalovirus (HCMV) ORF UL73 encodes the envelope glycoprotein gpUL73-gN, which shows seven genotypes (gN-1, gN-2, gN-3a, gN-3b, gN-4a, gN-4b, gN-4c). The goal of this study was to determine retrospectively the distribution of gN variants in solid organ transplant recipients with HCMV infection and to establish an association with parameters important for monitoring post-transplantation clinical course during a follow-up of up to 2 years. Peripheral blood leukocytes from 40 solid organ transplant recipients were analysed for pp65-antigen by immunofluorescence and gN genotyped by sequencing or RFLP analysis. A correlation between gN genotypes and antigenemia peak was found, showing a highly significant difference between gN-1 and gN-4b variants (P<0.005). In particular, gN-1 seems to be associated with patients developing low level antigenemia (<50 pp65-positive cells/2 x 10(5) PBLs; PPV = 90%), whereas gN-4b predicts significantly higher values (>50 pp65-positive cells/2 x 10(5) PBLs; PPV = 80%). Furthermore, the onset of positive antigenemia is significantly earlier in patients infected with a gN-4b strain, compared with those infected by a gN-1 variant. Reported data further support a role for gN genotypes in HCMV pathogenesis. gN-1 and gN-4b show a significantly different virulence and could serve as early predictors for the progression of HCMV infection in transplant patients.     

Soil-transmitted helminths and haemoglobin status among Afghan children in World Food Programme assisted schools

In recent years there have been major socio-economic changes within Afghanistan such that the present public health burden of soil-transmitted helminths (STH), especially that within school-aged children, remains to be determined. A baseline parasitological survey was therefore carried out in four defined areas of Afghanistan to better assess the distribution, prevalence and intensity of STH infections prior to a nationwide de-worming campaign beginning within World Food Programme assisted schools. A cross-sectional examination of 1001 children aged between 8 and 15 years old revealed that approximately half (47.2%) were infected with at least one STH. Infections with Ascaris lumbricoides were most widespread (40.9%) and elevated prevalences were detected in urban environments; for example, schoolchildren in Kabul were more likely to be infected (OR=2.2, 95% CI 1.6-3.0) than elsewhere and these infections were often of higher intensity (OR=7.6, 95% CI 4.9-11.8). Trichuris trichiura (9.9%) and hookworms (0.7%), previously unknown from Afghanistan, were encountered. The blood haemoglobin concentration of surveyed children was also assessed: 4% resulted to be anaemic (Hb<11 g dl(-1)), and 0.4% to be severely anaemic (Hb<7 g dl(-1)).     

The C2 variant of human serum transferrin retains the iron binding properties of the native protein

The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC 1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI--MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro 570 Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.     

Characterization of the c-Myb-responsive region and regulation of the human type I collagen alpha 2 chain gene by c-Myb

We have characterized the role of c-Myb and B-Myb in the regulation of human type I collagen alpha2 chain gene expression in fibroblastic cells. We have identified four Myb-binding sites (MBSs) in the promoter. Transactivation assays on wild type and mutant promoter-reporter constructs demonstrated that c-Myb, but not B-Myb, can transactivate the human type I collagen alpha 2 chain gene promoter via the MBS-containing region. Electrophoretic mobility shift assay experiments showed that c-Myb specifically binds to each of the four MBS; however, the mutagenesis of site MBS-4 completely inhibited transactivation by c-Myb, at least in the full-length promoter. In agreement with these results, c-myb(-/-) mouse embryo fibroblasts (MEFs) showed a selective lack of expression of type I collagen alpha 2 chain gene but maintained the expression of fibronectin and type III collagen. Furthermore, transforming growth factor-beta induced type I collagen alpha 2 chain gene expression in c-myb(-/-) MEFs, implying that the transforming growth factor-beta signaling pathway is maintained and that the absence of COL1A2 gene expression in c-myb(-/-) MEFs is a direct consequence of the lack of c-Myb. The demonstration of the importance of c-Myb in the regulation of the type I collagen alpha 2 chain gene suggests that uncontrolled expression of c-Myb could be an underlying mechanism in the pathogenesis of several fibrotic disorders.     

Enzymatic degradation and quantitative lectin labeling for characterizing glycoconjugates which act as lectin acceptors in cat submandibular gland

Summary Sites of binding of eight different lectins (LTA, UEA I, WGA, SBA, DBA, CON A, PNA, RCA I) to cat submandibular gland were studied after exposure of tissue sections to sialidase, -fucosidase, -galactosidase, -mannosidase, -N-acetylglucosaminidase. All lectins were affected by enzymatic predigestion and the labeling of individual lectins was highly dependent upon the glycosidase used to pretreat the sections. Glycoconjugates of demilunar, acinar and ductal cells exhibited a different composition of terminal sequences. For example, fucose proved to form the disaccharide fucose-galactose in demilunar and acinar cells, whereas it was present with the sequence fucose-N-acetyl-d-glucosamine in striated duct cells. Sialic acid participated both to the terminal sequence sialic acid-galactose and sialic acid-N-acetyl-d-galactosamine either in demilunar or in ductal cells. Lectin labeling combined with glycosidase digestion was also helpful in verifying the influence of neighbouring oligosaccharides on the affinity of lectins for the respective sugars.     

Drought effects on photosynthesis and fluorescence in hard wheat cultivars grown in the field

Net photosynthesis of the flag leaf of hard wheat ( Triticum durum L. evs Valforte, Produra, Adamello, Karel, Appulo and El Amel from the collection of the Instituto di Cerealicultura. Foggia, Italy) of different water potential has been studied on three consecutive years. Net photosynthesis was measured in natural conditions with a LI-COR portable instrument and in saturating CO₂ with an oxygen electrode. Net photosynthesis and stomatal conductance were significantly lower in the unirrigated leaves. However, the ratio of intercellular CO₂, concentration (C₁) to ambient CO₃ concentration (C_a) around the stressed plants was similar to the irrigated control. The maximal rate of photosynthesis in saturating CO₂, (P_nmax). measured in the second year of the experiment, was quite close to photosynthesis under natural conditions, indicating that CO₂ supply was not limiting. These results suggest that altered mesophyll photosynthetic capacity, rather than stomatal closure, causes the observed reduction in photosynthesis in the unirrigated plants. The variable fluorescence yield (_v/F_m) in predarkened leaves measured for two consecutive years, did not show differences between treatments or between cultivars. However, the analysis of the slow transients, measured the last year of the experiment, showed a linear relation between the fluorescence decline from the maximum initial level (P) and maximum photosynthesis (P_nmax).     

Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver

A trypsin-activated protein kinase has been isolated from rat liver using a peptide analogue of ribosomal protein S6 as a substrate in kinase assays. The structure of the peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, was based on a region of S6 containing both an insulin- and cyclic AMP-regulated phosphorylation site. The trypsin-activated protein kinase phosphorylated a corresponding site in the peptide analogue and ribosomal protein S6 that was distinct from the preferred site for cyclic AMP-dependent protein kinase. Ribosomal S6 contained at least one other major site for the trypsin-activated protein kinase.