Suppressive DNA vaccination in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis involves a T1-biased immune response

Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG(91-108) is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n)) rats. We examined the effects of DNA vaccines encoding MOG(91-108) in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-gamma, TNF-alpha, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.     

The new TNM classification of malignant melanoma

The identification of increasingly powerful prognostic factors has led to sequential modifications of the cutaneous melanoma staging system. The American Joint Committee on Cancer (AJCC) recently proposed major revision of tumor node metastasis (TNM) categories and stage groupings for melanoma. The authors summarize the main characteristics of this new TNM classification of malignant melanoma. The importance of the novel technique - sentinel node biopsy - in the management of malignant melanoma is discussed.     

The endogenous calcium ions of horseradish peroxidase C are required to maintain the functional nonplanarity of the heme

Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca(2+)-bound and Ca(2+)-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B(1g)- and B(2g)-type distortions in the Ca(2+)-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca(2+)-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion.     

Dominant species, functional assemblages and frequency of equilibrium phases in late summer phytoplankton assemblages in Hungarian small shallow lakes

Late summer phytoplankton associations were studied qualitatively and quantitatively in 80 Hungarian lakes altogether (mostly shallow salt lakes, reservoirs, oxbows, gravel pit lakes). Equilibrium phases sensu Sommer et al. (1993) were found only in 17 lakes. Most of them were under some kind (high salt content or very low level of nutrients) of stress factor. It is concluded that environmental stress forces phytoplankton communities towards equilibrium. No relationship between occurrence of equilibria and trophic state was found. Species number of non-equilibrated lakes was almost three times as high as those in equilibrium. Of the 31 recently described (Reynolds et al., 2002) phytoplankton assemblages most of those were recognized that are likely to occur in shallow lakes. Separation of a functional group W _S from W2 for Synura dominated lakes is suggested. It seemed also necessary to raise a group (Y _Ph) for lakes dominated by Phacotus. Sorting of Dinophyta species into different already described functional groups is desirable.     

Phosphoenolpyruvate-dependent tubulin-pyruvate kinase interaction at different organizational levels

Evidence for the direct binding of pyruvate kinase to tubulin/microtubule and for the inhibitory effect of phosphoenolpyruvate on tubulin-enzyme hetero-association were provided by surface plasmon resonance and pelleting experiments. Electron microscopy revealed that pyruvate kinase induces depolymerization of paclitaxel-stabilized microtubules into large oligomeric aggregates and bundles the tubules in a salt concentration-dependent manner. The C-terminal "tail"-free microtubules did not bind pyruvate kinase, suggesting the crucial role of the C-terminal segments in the binding of kinase. Immunoblotting and polymerization experiments with cell-free brain extract revealed that pyruvate kinase specifically binds to microtubules, the binding of pyruvate kinase impedes microtubule assembly, and phosphoenolpyruvate counteracts the destabilization of microtubules induced by pyruvate kinase. We also showed by immunostaining the juxtanuclear localization of pyruvate kinase in intact L929 cells and that this localization was influenced by treatments with paclitaxel or vinblastine. These findings suggest that the distribution of the enzyme may be controlled by the microtubular network in vivo.     

Evidence for microvascular dysfunction after prenatal dexamethasone at 0.7, 0.75, and 0.8 gestation in sheep

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM (n = 7) or saline (n = 7) was given starting at 103 days of gestation (dGA; term approximately 149 days). Fetal femoral arteries (approximately 300-microm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.     

SNPCEQer: detecting SNPs in sequences generated by the Beckman CEQ2000 DNA Analysis System

SNPCEQer identifies and reports SNPs in sequences obtained from the Beckman CEQ2000 DNA Analysis System. SNPCEQer aligns sequences obtained using CEQ2000 heterozygote detection analysis and reports discrepancies between individual sequences and the consensus sequence it generates from this set as SNPs when the individual base calls have high-quality values. SNPCEQer reported comparable numbers of SNPs to the UNIX-based PolyPhred (148 vs. 165, respectively) in regions amplified from eight genes. A total of 21 different SNPs was discovered. Each gene region was analyzed in 96-306 samples. SNPCEQer was designed to operate from Windows NT, making SNP detection more accessible to users without UNIX systems. SNPCEQer is available free of charge at http://innovation.swmed.edu.