Comparison of PSA-specific CD8<Superscript>+</Superscript> CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins

The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.     

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.     

Molecular characterization of the porcine testis-specificphosphoglycerate kinase 2 (<Emphasis Type="Italic">PGK2</Emphasis>) gene and its association with male fertility

We have isolated and characterized the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene, and 1665 bp of full-length PGK2 cDNA were also compiled using modified rapid amplification 5-RACE and 3-RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real-time quantitative PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis of a 10-month-old herniated boar and a very small amount of PGK2 expression in the testis of an 8–week-old cryptorchid piglet compared to an adult boar. Two SNPs in the PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser¹⁰²–Pro¹⁰²; SNP-B: Thr²⁶⁴–Lys²⁶⁴) were detected and genotyped among six pig breeds. The nucleotide C at SNP-A responsible for the amino acid exchange to proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between PGK2 genotypes and several traits of sperm quantity and quality were performed. The results showed that SNP-B has a positive significant effect on semen volume in the breed Pietrain (p=0.08), i.e., boars carrying genotype CC revealed an increased volume of 49 ml compared with boars having the genotype AA.The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AY500132 and AY486962.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.     

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.     

A combined treatment with 1alpha,25-dihydroxy-vitamin D3 and 17beta-estradiol reduces the expression of heat shock protein-32 (HSP-32) following cerebral cortical ischemia

1alpha,25-(OH)(2)-vitamin D(3) (1,25-D(3)) and 17beta-estradiol are both known to act neuroprotectively in certain experimental in vitro and in vivo settings and it has been noted that both steroids lead to an upregulation of certain neurotrophic factors. Here, we studied the effects of 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol or their combined application on heat shock protein-32 (HSP-32) distribution after focal cortical ischemia using the well established photothrombosis model. Heat shock protein-32 is a well-established marker of the cerebral oxidative stress response and contributes to neuroprotection by metabolising cytotoxic free heme to carbon monoxide, iron and biliverdin. Photothrombotically lesioned rats were injected i.p. 1h after injury with either 1 microg 1alpha,25-(OH)(2)-vitamin D(3)/kg or 7 microg 17beta-estradiol/kg or a combination of both steroids. Groups of non-lesioned steroid-treated rats and lesioned, solvent-treated rats served as controls. In contrast to non-lesioned rats, in lesioned animals a significant increase in heat shock protein-32 expression occurred which was slightly, but non-significantly altered in the groups treated either with 1alpha,25-(OH)(2)-vitamin D(3) or 17beta-estradiol alone when compared to the solvent-treated control group. Only the combined treatment with 1alpha,25-(OH)(2)-vitamin D(3) and 17beta-estradiol resulted in a significant reduction of glial heat shock protein-32 immunoreactivity within the lesion-remote cortical areas supplied by the affected middle cerebral artery (MCA), indicating that both steroids act synergistically in a protective manner.     

Phenotypical evidence for a gender difference in cardiac norepinephrine transporter function

Norepinephrine transporter (NET) function has a central role in the regulation of synaptic norepinephrine concentrations. Clinical observations in orthostatic intolerance patients suggest a gender difference in NET function. We compared the cardiovascular response to selective NET inhibition with reboxetine between 12 healthy men and 12 age-matched women. Finger blood pressure, brachial blood pressure, and heart rate were measured. The subjects underwent cardiovascular autonomic reflex testing and a graded head-up tilt test. In a separate study, we applied incremental concentrations of tyramine and isoproterenol through subcutaneous microdialysis catheters in eight men and in eight women. NET inhibition elicited a threefold greater increase in supine blood pressure in men than women (P < 0.05). The pressor response was driven by an increased cardiac output. The orthostatic heart rate increase during NET inhibition was greater in men than women (56 +/- 5 beats/min in men, 42 +/- 4 beats/min in women, P < 0.001). In contrast, NET inhibition resulted in a similar suppression in the cold pressor and handgrip response, low-frequency blood pressure oscillations, and venous norepinephrine in the supine position. Men and women were similarly sensitive to the lipolytic effect of isoproterenol and tyramine. We conclude that NET inhibition results in more pronounced changes in cardiac regulation in men than women. Our observations suggest that the NET contribution to cardiac norepinephrine turnover may be decreased in women. The gender difference in NET function may not be expressed in tissues that are less NET dependent than the heart.     

NMRShiftDB -- compound identification and structure elucidation support through a free community-built web database

Compound identification and support for computer-assisted structure elucidation via a free community-built web database for organic structures and their NMR data is described. The new database NMRShiftDB is available on . As the first NMR database, NMRShiftDB allows not only open access to the database but also open and peer reviewed submission of datasets, enabling the natural products community to build its first free repository of assigned 1H and 13C NMR spectra. In addition to the open access, the underlying database software is built solely from free software and is available under an open source license. This allows collaborating laboratories to fully replicate the database and to create a highly available network of NMRShiftDB mirrors. The database contains about 10,000 structures and assigned spectra, with new datasets constantly added. Its functionality includes (sub-) spectra and (sub-) structure searches as well as shift prediction of 13C spectra based on the current database material.     

Nose reconstruction by chondrocutaneous preauricular free flaps: anatomical basis and clinical results

Full-thickness defects of the nose result in considerable and distressing disfigurements. Ideally, reconstruction of such defects must be achieved in as few stages as possible and secondary, disfigurement kept to a minimum. In this study, the authors aimed to learn whether nose reconstruction could benefit from chondrocutaneous free flaps taken from the auricular tragus. In 72 ears, the vascular blood supply of the tragus was studied following injection of colored latex. Color-coded Duplex sonography served as a noninvasive method for demonstrating the blood supply of the target area. The procedure of nose reconstruction using the free chondrocutaneous tragus flap and the cosmetic results of this procedure in six patients are presented. Except for 2.8 percent of the anatomical specimens, the superficial temporal artery gave rise to the tragus and its overlying skin. The diameter of these branches ranged from 0.65 to 0.82 mm. Using the tragus composite free flap, the anatomical shape of the nose could be reconstructed successfully, and 6 months after surgery, the color and texture of the flap were very similar to those of the remaining nose. Using deeper parts of the tragus cartilage resulted in minimal scars and maintenance of the tragus anatomical shape. Free tragus flaps could be an alternative approach for nose reconstruction.