Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity induced by methamphetamine or MDMA

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), amphetamine derivatives widely used as recreational drugs, induce similar neurotoxic effects in mice, including a marked loss of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum. Although the role of dopamine in these neurotoxic effects is well established and pharmacological studies suggest involvement of a dopamine D2-like receptor, the specific dopamine receptor subtype involved has not been determined. In this study, we used dopamine D2 receptor knock-out mice (D2R(-/-)) to determine whether D2R is involved in METH- and MDMA-induced hyperthermia and neurotoxicity. In wild type animals, both drugs induced marked hyperthermia, decreased striatal dopamine content and TH- and DAT-immunoreactivity and increased striatal GFAP and Mac-1 expression as well as iNOS and interleukin 15 at 1 and 7days after drug exposure. They also caused dopaminergic cell loss in the SNpc. Inactivation of D2R blocked all these effects. Remarkably, D2R inactivation prevented METH-induced loss of dopaminergic neurons in the SNpc. In addition, striatal dopamine overflow, measured by fast scan cyclic voltammetry in the presence of METH, was significantly reduced in D2R(-/-) mice. Pre-treatment with reserpine indicated that the neuroprotective effect of D2R inactivation cannot be explained solely by its ability to prevent METH-induced hyperthermia: reserpine lowered body temperature in both genotypes, and potentiated METH toxicity in WT, but not D2R(-/-) mice. Our results demonstrate that the D2R is necessary for METH and MDMA neurotoxicity and that the neuroprotective effect of D2R inactivation is independent of its effect on body temperature.     

Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness affecting between 16 and 18 million people in North, Central, and South America for which no vaccine or satisfactory treatment is available (22). During its life cycle, the parasite goes through different stages in the vector (epimastigotes and metacyclic trypomastigotes) and in the mammalian host (amastigotes and bloodstream trypomastigotes). As part of its survival strategy in these varying environments, the parasite has developed a large repertoire of multigene families (9, 11, 12, 16). Among these families, the dispersed gene family 1 (DGF-1) has approximately 565 copies, ranging from 6 to 10 kbp, dispersed throughout the parasite genome (11). The members of the DGF-1 family encode proteins that share 85 to 95% sequence identity (11). Wincker and colleagues first identified clones bearing a common repeated sequence from a T. cruzi genome library (24) and later described the nucleotide sequence of a representative gene (DGF-1.1) (23). They concluded, from in silico studies, that DGF-1 genes encoded putative cell surface proteins (23). In 2005, Kim and colleagues (16) described a new member of this family (DGF-1.2) located in the subtelomeric region of a T. cruzi chromosome surrounded by mainly two kinds of sequences: genes encoding the trans-sialidase (TS) and retrotransposon hot spot (RHS) protein families. The sizes of the open reading frames (ORFs) of DGF-1 genes and their abundance in the T. cruzi genome suggested that they are essential sequences for parasite survival. Furthermore, the existence of some telomeric DGF-1 copies that were always flanked by pseudogenes suggested that these genes have been subjected to strong selective pressure and, as a consequence, that they should be expressed at some life cycle stage of the parasite (16).A glycopeptide shared by several members of the DGF-1 family was recently detected in a glycoproteomic study of T. cruzi trypomastigotes, demonstrating that at least a DGF-1 family member protein is actually expressed and N-glycosylated (3). We also detected a number of peptides corresponding to several DGF-1 family member proteins in a proteomic study of acidocalcisome fractions of epimastigotes of T. cruzi (R. Docampo, J. A. Atwood, R. Tarleton, and R. Orlando, unpublished data). However, this family of proteins has no known orthologs in other species, even in trypanosomatids, and little is known about their localization, expression patterns, and functions in T. cruzi.In the present work, we prepared affinity-purified antibodies against a peptide of the DGF-1.2 protein and investigated its subcellular localization by immunofluorescence microscopy. We also used mass spectrometry (MS) to identify specific peptides recognized by anti-DGF-1.2 antibodies by using fingerprinting analysis.We found that the antibodies preferentially labeled amastigotes. The localization of the protein was in intracellular bodies and not on the cell surface and changed during amastigote development. During the in vitro trypomastigote-to-amastigote transition, we detected continuous secretion of DGF-1.2 into the medium, peaking at 14 h. Anti DGF-1 antibodies that recognized the intracellular protein in both differentiation forms also recognized the secreted protein from trypomastigotes and amastigotes. Finally, when epimastigotes were subjected to starvation, there was a decrease in labeling of the cells and the appearance of defined bands of smaller molecular mass in Western blots, suggesting its cleavage.     

Stereoselective synthesis by olefin metathesis and characterization of η-carotene (7,8,7',8'-tetrahydro-β,β-carotene)

The purported structure of the elusive η-carotene (7,8,7',8'-tetrahydro-β,β-carotene), a natural C(40) carotenoid first detected in the berries of Lonicera japonica and in citrus fruits sixty years ago, has been synthesized by olefin cross-metathesis/dimerization of a C(21) polyene derived from trans-7,8-dihydroretinal, thus allowing the full characterization of this highly unstable natural product.     

Spontaneous in vitro maturation of oocytes prior to ovarian tissue cryopreservation in natural cycles of oncologic patients

Purpose

To evaluate the recovery rate and spontaneous in vitro maturation (IVM) of immature oocytes enclosed within or released from follicles during the processing of ovarian tissue prior to its cryopreservation.

Methods

Thirty-three oncologic patients who had not previously undergone chemo or radiotherapy underwent ovarian tissue cryopreservation (OTC) during natural menstrual cycles. Immature oocytes, enclosed within follicles or released during ovarian cortex processing, were collected and matured spontaneously in vitro for 48 h. Nuclear maturation was assessed every 24 h and the ability of the IVM oocytes to display a normal activation response following parthenogenetic activation was evaluated. The following outcome measures were also evaluated: disease, age, FSH, LH, E2, P4 and AMH serum levels, menstrual cycle day, recovery and spontaneous IVM and parthenogenetic activation rates.

Results

Oocytes recovered per patient were 3.3 ± 0.7 (1.8–4.7 oocytes, 95CI), regardless of the menstrual phase. The mean number of IVM oocytes per patient was 1.3 ± 0.2 oocytes (95CI: 0.8–1.8), regardless of menstrual phase (p = 0.86) and oocyte origin (p = 0.61). Forty-one percent of oocytes extruded the second polar body and formed one pronucleus after parthenogenetic activation.

Conclusion

Twenty-one of the 33 women (63.6 %) requesting OTC produced at least one mature oocyte.     

Reproducibility and clinical relevance of the ocular response analyzer in nonoperated eyes: corneal biomechanical and tonometric implications

PURPOSE: To assess the reproducibility of the ocular response analyzer (ORA) in nonoperated eyes and the impact of corneal biomechanical properties on intraocular pressure (IOP) measurements in normal and glaucomatous eyes. METHODS: In the reliability study, two independent examiners obtained repeated ORA measurements in 30 eyes. In the clinical study, the examiners analyzed ORA and IOP-Goldmann values from 220 normal and 42 glaucomatous eyes. In both studies, Goldmann-correlated IOP measurement (IOP-ORAg), corneal-compensated IOP (IOP-ORAc), corneal hysteresis (CH), and corneal resistance factor (CRF) were evaluated. IOP differences of 3 mm Hg or greater between the IOP-ORAc and IOP-ORAg were considered outcome significant. RESULTS: Intraexaminer intraclass correlation coefficients and interexaminer concordance correlation coefficients ranged from 0.78 to 0.93 and from 0.81 to 0.93, respectively, for all parameters. CH reproducibility was highest, and the IOP-ORAg readings were lowest. The median IOP was 16 mm Hg with the Goldmann tonometer, 14.5 mm Hg with IOP-ORAg (P < 0.001), and 15.7 mm Hg with IOP-ORAc (P < 0.001). Outcome-significant results were found in 77 eyes (29.38%). The IOP-ORAc, CH, and CRF were correlated with age (r = 0.22, P = 0.001; r = -0.23, P = 0.001; r = -0.14, P = 0.02, respectively), but not the IOP-ORAg or IOP-Goldmann. CONCLUSIONS: The ORA provides reproducible corneal biomechanical and IOP measurements in nonoperated eyes. Considering the effect of ORA, corneal biomechanical metrics produces an outcome-significant IOP adjustment in at least one quarter of glaucomatous and normal eyes undergoing noncontact tonometry. Corneal viscoelasticity (CH) and resistance (CRF) appear to decrease minimally with increasing age in healthy adults.     

Glaucoma probability score vs Moorfields classification in normal, ocular hypertensive, and glaucomatous eyes

PURPOSE: To evaluate the Heidelberg Retina Tomograph III (HRT III) glaucoma probability score in differentiating normal from pathologic eyes and to compare the glaucoma probability score with Moorfields regression analysis (MRA). DESIGN: Prospective cross-sectional study. METHODS: Fifty-nine normal, 40 hypertensive, and 83 glaucomatous eyes were examined with Swedish interactive threshold algorithm standard 24-2 visual fields and HRT III. Sensitivity and specificity were evaluated using global and sectorial glaucoma probability score and MRA compared with damage in visual fields. Areas under receiver operating characteristic (ROC) curves were evaluated. Agreement between MRA and glaucoma probability score was calculated using the kappa coefficient. Glaucoma probability score was considered to be displaced when a symbol was outside and the opposite symbol was inside the optic disk. RESULTS: MRA sensitivity and specificity were 39.8% and 93.2% (most specific criteria) and 68.7% and 83.1% (least specific criteria), respectively. Glaucoma probability score sensitivity and specificity were 71.1% and 69.5% (most specific criteria) and 85.5% and 54.2% (least specific criteria), respectively. Visual field parameters were related to the global (P = .001) and sectorial (P < .05) glaucoma probability score. A displaced glaucoma probability score was found in 35 eyes, but with unchanged glaucoma probability score sensitivity and specificity. Areas under the ROC curves of glaucoma probability score was 0.77. The kappa coefficient was 0.34. CONCLUSIONS: Glaucoma probability score analysis tends to be more sensitive but less specific than MRA. Glaucoma probability score did not differentiate normal and hypertensives eyes. When displaced, glaucoma probability score sensitivity and specificity were unchanged. MRA and glaucoma probability score agreement was low. Glaucoma probability score is advantageous over MRA in early-stage glaucoma.     

Coronary Involvement in Infants with Kawasaki Disease Treated with Intravenous γ-Globulin

Aim To analyze the clinical spectrum and the incidence of coronary involvement in infants with typical Kawasaki's disease (KD). Patients and Methods A retrospective study was performed on children one year of age or younger diagnosed from February 1992 to January 2006 with typical KD. Children with incomplete forms of the disease were not included. Results Twenty-five infants were diagnosed with KD during the study period. The median age of the patients was 10 months (range, 4–12 months). All children but one received intravenous gammaglobulin (IVIG), 84% before the 10th day of disease. Seven patients (28%) required the administration of more than one dose of IVIG, because persistence of fever. Coronary artery disease (CAD) was recorded in 6 cases (24%), five of them being boys. All patients with CAD were treated with ASA plus IVIG and 84% of them received this therapy within the first 10 days of the KD onset. Conclusions In spite of the exclusion of our study of incomplete presentations and of an early administration of IVIG in our patients, we have observed a high rate of infants who developed CAD, which is similar to the one reported in children who do not receive IVIG.