Protection of atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination

Although vaccines against infectious pancreatic necrosis (IPN) based on inactivated virus or recombinant structural viral proteins are commercially available, the protection is not complete and the disease is still a problem for the Atlantic salmon Salmo salar farming industry. In the present study, 5 different plasmids that expressed whole or parts of the large open reading frames (ORF) of Segment A of the IPN virus (IPNV) were constructed. The plasmids were shown to express proteins in cell cultures and in zebrafish Danio rerio in vivo. The specificities of the expressed proteins were confirmed by staining with IPNV-specific monoclonal antibodies (MAb) The plasmids were then used alone or in different combinations to vaccinate groups of Atlantic salmon, which subsequently were challenged in an experimental assay for IPN. A high level of protection was induced only by the plasmid combination that contained a plasmid expressing all the large ORF polyprotein.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Fine needle aspiration cytology of neurothekeoma. A case report

BACKGROUND: Neurothekeoma (NT) is a rare, benign neoplasm of soft parts with a distinctive histologic appearance. To our knowledge, the cytologic findings have not been described before. We present a case of NT with the cytologic features on fine needle aspiration cytology (FNAC). CASE: A 54-year-old female presented with a circumscribed nodule in the left breast. The lesion was evaluated by FNAC. The smears showed an abundant, metachromatic, myxoid matrix with fusiform and epithelioid cells, some binucleated or multinucleated, loose or in groups and sometimes forming concentric whorls. The lesion was removed, and the diagnosis of NT was made after histopathologic study. CONCLUSION: NT is an extremely rare neoplasm in the mammary region. Fusiform and epithelioid cells arranged in concentric whorls in a myxoid tumor of soft tissue are a distinctive characteristic of this neoplasm and can suggest the diagnosis.     

De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase-1 expression and fatty acid metabolism in liver of fenofibrate-treated rats

Clavaminate synthase (CAS), a 2-oxoglutarate (2OG) dependent dioxygenase, catalyses three steps in the biosynthesis of clavulanic acid. Crystals of CAS complexed with Fe(II), 2OG and deoxyguanidinoproclavaminate were exposed to nitric oxide (NO) acting as a dioxygen analogue. Prior to exposure with NO, the active site Fe(II) is octahedrally coordinated by a water molecule, the 2-oxo and 1-carboxylate groups of 2OG, and the side-chains of an aspartyl and two histidinyl residues. NO binds to the position previously occupied by the 2OG 1-carboxylate concomitant with rearrangement of the latter to the position previously occupied by the displaced water.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Purification,cofactor analysis,and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting

Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.