Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C)

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.     

Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry

We describe methods for mass spectrometric identification of heme-containing peptides from c-type cytochromes that contain the CXXCH (X=any amino acid) sequence motif. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference for both 2+ and 3+ peptide charge states; both sequence and charge affect the extent of heme loss. Application to Shewanella oneidenis demonstrated the utility of this approach for identifying c-type heme-containing peptides from complex proteome samples.     

Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal

The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation. Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner. To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized. Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes. Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT. In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites. Retrotransposons with mutations in each of these sites have transposition defects. Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies. Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa. Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first. Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway.     

Investigation of chromatographic conditions for the separation of cefuroxime axetil and its geometric isomer

The optimal chromatographic conditions for the separation of the syn- and anti-geometric isomers of cefuroxime axetil applying RP-HPLC and micellar liquid chromatography (MLC) methods were investigated. The possibility to separate diastereoisomers of syn- and anti-cefuroxime axetil was observed. Investigations were performed using three columns, two classical silicas and one with hybrid particle technology. Three aqueous-organic and one micellar mobile phases were used. The best results were achieved using micellar mobile phase. Optimization study was performed using different micellar mobile phases. MLC method is sensitive and applicable in purity and stability testing.     

The Introduced Green Alga Caulerpa Taxifolia Continues to Spread in the Mediterranean

The tropical green alga Caulerpa taxifolia in the Mediterranean has spread steadily since its introduction in 1984. At the end of 2000, approximately 131km² of benthos had been colonized in 103 independent areas along 191km of coastline in six countries (Spain, France, Monaco, Italy, Croatia and Tunisia). Large regions neighboring the invaded areas appear favorable to further colonization, and there is thus no reason to believe that spreading will slow down in the years to come.     

Relationship between the effectiveness of the antitumor action of sarcolysine and the latency period between transplantation and tumor appearance

Sarcoma induced with 3-methylcholantrene in mice was transplanted to syngeneic recipients. The latent period of the tumour growth varied from 7 to 26 days. Sarcolysine was injected once when the tumour became palpable. When the tumours were revealed 7 to 8 and 10 to 15 days after the inoculation, the tumour growth inhibition was accompanied by increased survival of mice, particularly marked at the latter period. When the tumours were revealed 17 to 20 or 22 to 26 days after the transplantation, inhibition of the tumour growth was not accompanied by any increase of survival.     

A Rapid and Simple Method for DNA Engineering Using Cycled Ligation Assembly

DNA assembly techniques have developed rapidly, enabling efficient construction of complex constructs that would be prohibitively difficult using traditional restriction-digest based methods. Most of the recent methods for assembling multiple DNA fragments in vitro suffer from high costs, complex set-ups, and diminishing efficiency when used for more than a few DNA segments. Here we present a cycled ligation-based DNA assembly protocol that is simple, cheap, efficient, and powerful. The method employs a thermostable ligase and short Scaffold Oligonucleotide Connectors (SOCs) that are homologous to the ends and beginnings of two adjacent DNA sequences. These SOCs direct an exponential increase in the amount of correctly assembled product during a reaction that cycles between denaturing and annealing/ligating temperatures. Products of early cycles serve as templates for later cycles, allowing the assembly of many sequences in a single reaction. To demonstrate the method’s utility, we directed the assembly of twelve inserts, in one reaction, into a transformable plasmid. All the joints were precise, and assembly was scarless in the sense that no nucleotides were added or missing at junctions. Simple, efficient, and low-cost cycled ligation assemblies will facilitate wider use of complex genetic constructs in biomedical research.     

<Emphasis Type="Italic">Castanea sativa</Emphasis>: genotype-dependent recovery from chestnut blight

In Lovran (coastal Croatia), a unique forest/orchard of evenly mixed grafted marrons and naturally growing nongrafted sweet chestnut trees exists. This old chestnut population has been devastated by chestnut blight, caused by an aggressive introduced pathogenic fungus, Cryphonectria parasitica. However, initial observations indicated recovery of naturally growing chestnut trees in that area, mediated by Cryphonectria-associated hypovirus (Cryphonectria hypovirus 1 (CHV-1)). Such recovery was not observed on grafted trees. Genotyping both, we confirmed the clonal origin of the grafted ones—marrons. No significant difference was observed between fungal strains isolated from naturally growing trees and the ones from marrons regarding fungal vegetative compatibility types or the prevalence of CHV-1. A strong correlation was observed between the types of canker: active/deep-expanding versus healing callus or superficial necrosis and the absence or presence of CHV-1 in the fungal isolates, sampled from naturally growing trees (Spearman rho 0.686, p value 7.81?×?10^?5, Kendall tau 0.686, p value 5.18?×?10^?7). Such correlation was not observed on marrons (Spearman rho 0.236, p value 0.235, Kendall tau 0.236, p value 0.084), because, unexpectedly, active/deep-expanding cankers were often associated with hypovirulent fungal isolates. These data indicate that the lack or unequal distribution of naturally occurring hypovirulence were not the cause of substantial marron decay in Lovran. Ecological and age-dependant differences were ruled out because all sampled trees are growing in close proximity and are of similar age. The results imply that the marron genotype is especially vulnerable and its ability to recover is limited even when the hypovirulent strain of the fungus is present in the canker.     

The role of IgG avidity determination in diagnosis of Epstein-Barr virus infection in immunocompetent and immunocompromised patients

There is a high degree of variability in the serologic response to Epstein-Barr virus (EBV) infection, especially in viral capsid antigen (VCA)-IgM antibodies. Therefore, additional tests are needed to confirm primary infection. We evaluated the value of IgG avidity determination in diagnosis of EBV infection in immunocompetent and immunocompromised patients. A total of 236 serum samples from immunocompetent patients with symptoms suggestive of EBV infection were tested for the presence of VCA-IgM/IgG antibodies and IgG avidity. Using IgG avidity, acute primary infection was confirmed in 56.7% of the immunocompetent patients with positive and in 1.8% of patients with negative VCA-IgM. Recent primary infection was documented in 8.9% of the IgM positive and 3.5% of the IgM negative patients. In patients with indeterminate serology (equivocal IgM), 6.7% were classified by avidity index (AI) as acute primary infection, 10.0% as post-acute and 83.3% as past infection cases. Concerning the 32 immunocompromised patients, recent primary infection was documented in 3 of the 14 IgM positive patients. High AI was detected in 11 of these patients, indicating an IgM response due to reactivation. Determination of IgG avidity in combination with classical serologic markers seems to be a reliable method to confirm primary infection both in immunocompetent and immunocompromised patients. It may be especially useful to differentiate cases of primary infection in patients with undetectable VCA-IgM antibodies or indeterminate routine EBV serology.     

Galectin-1 is part of human trophoblast invasion machinery--a functional study in vitro

Background

Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.

Methods and Findings

Function blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM±6.3, P<0.001) and to 66% of control (SEM±1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM±16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM±51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM±58, P<0.001) by CS-gal-1, and to 200% (SEM±24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.

Conclusion and Significance

These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.