Linking DJ-1 to neurodegeneration offers novel insights for understanding the pathogenesis of Parkinson’s disease

Rare monogenic forms of Parkinson's disease (PD) are promoting our understanding of the molecular pathways involved in the common, non-Mendelian forms of the disease. Here, we focus on PARK7, an autosomal recessive form of early-onset parkinsonism caused by mutations in the DJ-1 gene. We first review the genetics of this form and the rapidly expanding knowledge about the structure and biochemical properties of the DJ-1 protein. We also discuss how DJ-1 dysfunction might lead to neurodegeneration, and the implications of this novel piece of information for the pathogenesis of the common PD forms. Although much work remains to be done to clarify the biology of DJ-1, its proposed activity as a molecular chaperone and/or as oxidative sensor appear intriguing in the light of the current theories on the pathogenesis of PD.     

Chronic myelogenous leukemia with a complex Ph1 translocation in an XYY male

We encountered a 38-year-old Japanese male patient with chronic myelogenous leukemia (CML), whose bone marrow and peripheral blood cells during the chronic and blastic phases contained a complex Ph¹ translocation and an extra Y chromosome [i.e., 47,XYY,t(9;22;13)(q34;q11;q14)]. A karyotypic analysis of PHA-stimulated lymphocytes showed the constitutional karyotype to be 47,XYY. Thus, it was considered that CML with a complex Ph¹ translocation developed in an XYY male; such a case has not been reported, so far. A B-lymphocyte cell line with the complex Ph¹ translocation was established by the procedure of Epstein-Barr virus transformation. The presence of the complex Ph¹ translocation in the B-lymphocyte cell line suggests that some of the B lymphocytes in this patient originated from the CML clone.     

Egfl7 knockdown causes defects in the extension and junctional arrangements of endothelial cells during zebrafish vasculogenesis.

The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.     

Monoclonal antibodies to the human mammary gland: III. Monoclonal antibody LICR-LON-M18 identifies impaired expression and excess sialylation of the I(Ma) cell-surface antigen by primary breast carcinoma cells

Monoclonal antibody LICR -LON- M18 identifies the immunodominant oligosaccharide sequence of the I(Ma) blood-group antigen: Gal beta 1----4GlcNAc beta 1----6--. In primary breast cancers this structure is almost totally cryptic, due to "masking" by sialic acid, but can be revealed by digestion with the specific glycosidase neuraminidase. Following desialylation, light microscopic immunohistochemical examination has revealed the epitope identified by LICR -LON- M18 to be heterogeneously distributed throughout the population of breast carcinoma cells. These tumor cells express the antigen as both a cytoplasmic and a surface membrane determinant. In the normal human breast, this structure is expressed exclusively along the luminal plasma membranes of the duct and alveolar littoral epithelial cells. Desialylation of tissue sections of normal resting and lactating breast epithelium with neuraminidase virtually abolishes the heterogeneous intercellular distribution of the I(Ma) determinant. In desialylated nonneoplastic breast tissues, the expression of this antigen is observed within the cytoplasm of some myoepithelial cells, but not in the littoral epithelial cells. The expression of the I(Ma) antigen by neoplastic and normal breast epithelial cells has also been compared with that of the oligosaccharide sequence Gal beta 1----3GalNAc. This structure, recognized by peanut agglutinin, forms the dominant portion of the Thomsen-Friedenreich antigen. With respect to normal and lactating breast epithelial cells, both oligosaccharide structures are sialylated and appear to be similarly misprocessed by breast carcinomas. The masking of surface carbohydrate determinants and the faulty processing of structures usually expressed on the surface of non-neoplastic breast epithelial cells may be important phenomena in the pathobiology of breast carcinomas.     

A small-conductance charybdotoxin-sensitive,apamin-resistant Ca2+-activated K+ channel in aortic smooth muscle cells (A7r5 line and primary culture)

A small conductance K⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5 and also in rat aortic smooth muscle cells in primary culture, using conventional single-channel recording techniques. The single-channel conductance shows no rectification, either in the range –70 to +40 mV under asymmetrical conditions (9.1 pS), or in the range –100 to +50 mV in symmetrical 150 mM K⁺ (37 pS). Channel activity is reversibly inhibited by extracellular application of charybdotoxin, with a concentration of 8 nM producing half-maximal inhibition. It is unaffected by apamin or scyllatoxin. Channel activity depends on the presence of free Ca²⁺ on the cytosolic face of the membrane, with an activation zone between 0.1 and 1 M. This small-conductance, charybdotoxin-sensitive, Ca²⁺-regulated K⁺ channel is activated by vasoconstrictors such as vasopressin and endothelin.     

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.     

Increased fluidity of Plasmodium berghei-infected mouse red blood cell membranes detected by electron spin resonance spectroscopy

The fluidity of Plasmodium berghei-infected mouse red cell membranes is increased over that of uninfected cells at both 24°C and 37°C. This was demonstrated by electron spin resonance spectroscopy using the hydrocarbon spin labels 2-dodecyl-2′,5,5′-trimethyloxazolidine-N-oxyl and 2-heptyl-2′ -hexyl-5,5′-dimethyloxazolidine-N-oxyl to label regions of the bilayer near its surface, and deeper within the hydrocarbon region, respectively. Arrhenius plots of the ‘empirical motion parameter’ (R_i) obtained from 2-heptyl-2′-hexyl-5,5′-dimethyloxazolidine-N-oxyl-labeled cells versus temperature over the range from 0 to 45°C showed an hysteretic behavior of the spin labels in the membranes of both mature and immature uninfected cells. Such hysteretic behavior was consistently lacking in membranes of infected cells. These differences in membrane fluidity and spin label behavior are interpreted to reflect biochemical modifications of the red cell membrane which occur with infection by the malarial parasite.     

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.     

Gamma delta T cells expressing CD8 or CD4low appear early in murine foetal thymus development.

Three-colour flow cytometry was used to study the distribution of TCR gamma delta+ cells among CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- cell populations during thymic development. Thymocytes were obtained either directly from embryos at different stages of gestation (ex vivo) or from organ cultures maintained in vitro. In both cases, TCR gamma delta+ cells were found predominantly among the double negative (CD4-CD8-) and CD8 single positive subsets. These cells were actively dividing as demonstrated by 7 amino actinomycin D (7AAD) labelling. A small population of TCR gamma delta+ cells expressing low levels of CD4 was identified early and transiently (days 15-18) during development, but this subset was rare in the adult thymus. In newborn mice, adult mice, and late during organ culture, TCR gamma delta+ cells were found mainly within the CD4-CD8- compartment of thymocytes, although a minor population of CD8+ cells (5-10%) bearing gamma delta receptor was routinely observed. In contrast, few gamma delta cells were contained among the CD4+CD8+ subset at any timepoint studied. These data highlight differences between the ontogeny of alpha beta and gamma delta cells in the thymus, and suggest that a CD4+CD8+ intermediate may not be a requisite for the intrathymic differentiation of murine gamma delta T cells.     

Interactions of the enantiomers of 3-O-methyldobutamine with α- and β-adrenoceptors in vitro

Summary The enantiomers of 3-O-methyldobutamine, a metabolite of dobutamine, were evaluated for their - and -adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in isolated guinea pig aorta. However, both enantiomers of 3-O-methyldobutamine were competitive ₁-adrenoceptor antagonists, with the (+)-enantiomer being approximately 10-fold more potent than the (-)-enantiomer as assessed either in guinea pig aorta or by displacement of ³H-prazosin binding from ₁-adrenoceptors in rat cerebral cortex. The ₁-adrenoceptor blocking activity of (+)-3-O-methyldobutamine was relatively potent and corresponded to a pA₂ of 7.33 in guinea pig aorta and a-log K _i of 7.72 in radioligand binding studies. Neither enantiomer of 3-O-methyldobutamine possessed ₂-adrenoceptor agonist activity in field-stimulated guinea pig ileum. Although (+)-3-O-methyldobutamine weakly inhibited the twitch response in field-stimulated guinea pig ileum, the response was not blocked by the selective ₂-adrenoceptor antagonist, yohimbine, and was found to result from weak anticholinergic activity (pA₂=5.06). Neither enantiomer of 3-O-methyldobutamine possessed ₁-adrenoceptor agonist activity in guinea pig atria, however the (+)-enantiomer was a weak noncompetitive antagonist at ₁-adrenoceptors. In contrast, both enantiomers of 3-O-methyldobutamine were weak ₂-adrenoceptor agonists in rat uterus, however these weak effects were not highly stereoselective, which was also confirmed in radioligand binding studies. The results of the present study indicate that 3-O-methyldobutamine is a potent and highly selective ₁-adrenoceptor antagonist, with minimal activity at ₂-, ₁- and ₂-adrenoceptors. It is hypothesized that the potent ₁-adrenoceptor antagonist activity of 3-O-methyldobutamine, which resides predominantly in the (+)-enantiomer, may play a role in the hemodynamic effects of dobutamine, by contributing, in part, to the decrease in total peripheral vascular resistance observed following administration of dobutamine.