Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Oxytocin: the neuropeptide of love reveals some of its secrets

The neuropeptide oxytocin is synthesized in the brain and released from neurohypophyseal terminals into the blood and within defined brain regions that regulate emotional, cognitive, and social behaviors. A recent study of CD38-/- mice (Jin et al., 2007) has demonstrated an essential role for the transmembrane receptor CD38 in secretion of oxytocin into the blood.     

Strong seasonal differences of bacterial polysaccharide utilization in the North Sea over an annual cycle

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes – extracellular hydrolysis and selfish uptake – have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved.     

Lead resistance and sensitivity in Staphylococcus aureus

Abstract Five lead-resistant strains of Staphylococcus aureus were isolated. Plasmid-free lead-sensitive variants were obtained from the three plasmid-bearing strains. Lead-resistant strains tolerated an approximately 600 × higher Pb(NO₃)₂ concentration than lead-sensitive strains. Both types of strains initially bound lead, but only the resistant strains accumulated the metal as an intracellular lead-phosphate.     

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.     

alpha1-antitrypsin and its C-terminal fragment attenuate effects of degranulated neutrophil-conditioned medium on lung cancer HCC cells, in vitro

BACKGROUND: Tumor microenvironment, which is largely affected by inflammatory cells, is a crucial participant in the neoplastic process through promotion of cell proliferation, survival and migration. We measured the effects of polymorphonuclear neutrophil (PMN) conditioned medium alone, and supplemented with serine proteinase inhibitor alpha-1 antitrypsin (AAT) or its C-terminal fragment (C-36 peptide), on cultured lung cancer cells. METHODS: Lung cancer HCC cells were grown in a regular medium or in a PMN-conditioned medium in the presence or absence of AAT (0.5 mg/ml) or its C-36 peptide (0.06 mg/ml) for 24 h. Cell proliferation, invasiveness and release of IL-8 and VEGF were analyzed by [3H]-thymidine incorporation, Matrigel invasion and ELISA methods, respectively. RESULTS: Cells exposed to PMN-conditioned medium show decreased proliferation and IL-8 release by 3.9-fold, p < 0.001 and 1.3-fold, p < 0.05, respectively, and increased invasiveness by 2-fold (p < 0.001) compared to non-treated controls. In the presence of AAT, PMN-conditioned medium loses its effects on cell proliferation, invasiveness and IL-8 release, whereas VEGF is up-regulated by 3.7-fold (p < 0.001) compared to controls. Similarly, C-36 peptide abolishes the effects of PMN-conditioned medium on cell invasiveness, but does not alter its effects on cell proliferation, IL-8 and VEGF release. Direct HCC cell exposure to AAT enhances VEGF, but inhibits IL-8 release by 1.7-fold (p < 0.001) and 1.4-fold (p < 0.01) respectively, and reduces proliferation 2.5-fold (p < 0.01). In contrast, C-36 peptide alone did not affect these parameters, but inhibited cell invasiveness by 51.4% (p < 0.001), when compared with non-treated controls. CONCLUSIONS: Our data provide evidence that neutrophil derived factors decrease lung cancer HCC cell proliferation and IL-8 release, but increase cell invasiveness. These effects were found to be modulated by exogenously present serine proteinase inhibitor, AAT, and its C-terminal fragment, which points to a complexity of the relationships between tumor cell biological activities and local microenvironment.     

Identification of Genes Essential for Growth at High Salt Concentrations Using Salt-Sensitive Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 6803

A collection of 17 salt-sensitive mutants of the cyanobacterium Synechocystis sp. strain PCC 6803 was obtained by random cartridge mutagenesis. The genes coding for proteins essential for growth at high salt concentrations were mapped on the completely known genome sequence of this strain. The two genes coding for enzymes involved in biosynthesis of the osmolyte glucosylglycerol were affected in nine mutants. Two mutants defective in a glycoprotease encoding gene gcp showed a reduced salt resistance. Four genes were identified not previously known to be essential for salt tolerance in cyanobacteria. These genes (slr1799, slr1087, sll1061, and sll1062) code for proteins not yet functionally characterized. Received: 21 May 2001 / Accepted: 27 June 2001