Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

1. Download : Download high-res image (63KB)
2. Download : Download full-size image

Highlights

• •MS-based clinical assay that accurately determines phospho Rab10 occupancy.
• •Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.
• •Determination of pRab levels in neutrophils of Parkinson disease patients.
• •Relevance of pRab levels as marker of PD.

     

Evaluation of the efficacy of various hypotensive drugs in broad-breasted white turkeys as an experimental model of arterial hypertension with high catecholamine levels

Male Broad Breasted White Turkeys (BBWT) represent an experimental model of arterial hypertension characterized by high levels of circulating and tissue catecholamines. We thought interesting to evaluate the efficacy of the long term treatment with different antihypertensive drugs. 59 male BBWT were studied, divided in five groups. The first group (13 animals) was treated with placebo; the second (14 animals) with oxprenolol 4 mg/Kg/q.d.; the third (10 animals) with labetalol 25 mg/Kg/q.d.; the fourth (11 animals) with verapamil 15 mg/Kg/ q.d.; the fifth (11 animals) with captopril 8 mg/Kg/ q.d. and furosemide 2,5 mg/Kg/q.d. All drugs were given p.o, from the 8th to the 33rd week of age. Weekly or every two weeks Blood Pressure (BP) and Heart Rate (HR) were measured by an indirect method. In all animals BP progressively increased and HR progressively decreased with age. Only the labetalol-treated animals showed a significant reduction of BP and HR through the study period as compared with the placebo-treated animals. These results confirm the preminent role played by the high levels of circulating catecholamines in determining and maintaining the arterial hypertension.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture

Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells     

Expression of HOX homeogenes in human neuroblastoma cell culture lines

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.     

Correlation between Lack of Receptacle Growth in Response to Auxin and Accumulation of a Specific Polypeptide in a Strawberry (Fragaria ananassa Duch.) Variant Genotype

Receptacle growth in strawberry (Fragaria ananassa Duch. cv.Ozark Beauty) occurred after either pollination or auxin treatment.In a strawberry variant genotype (Washington State UniversitySelection No. 12/13), pollination did not lead to receptaclegrowth but application of -naphthaleneacetic acid (NAA) at anthesisresulted in normal receptacle growth. The receptacles of OzarkBeauty retained their ability to respond to auxin at least upto 36 days after anthesis. However, delay of auxin applicationto the receptacles of the variant genotype resulted in decreasedauxin-responsive growth and auxin application after the 10thday of anthesis led to very little growth. The loss of auxin-responsivegrowth of the receptacle of the variant genotype was not associatedwith any loss of auxin binding activity of receptacle membranes.If auxin was not supplied to the receptacles of the variantgenotype at anthesis, the receptacles did not grow and a polypeptideof 52,000 M_r accumulated. Application of NAA to the receptaclesof the variant genotype at anthesis or on the fifth day afteranthesis resulted in the growth of the receptacle and the 52,000M_r polypeptide did not accumulate. Application of NAA to thereceptacles of the variant genotype on the 10th or the 15thday after anthesis led to very little growth of the receptacleand the 52,000 M_r polypeptide accumulated to high levels. Theseresults suggested a correlation between the lack of receptaclegrowth in response to auxin and accumulation of the 52,000 M_rpolypeptide. ¹ Current adress: The Institute of Applied Research, Ben GurionUniversity of the Negev, Beer-Sheva, Israel. (Received August 6, 1984; Accepted December 11, 1984)     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.     

Costs and benefits of maternal aggression in lactating female rhesus macaques

Data from over 400 hr of observation of mother-infant rhesus macaques indicate that during the first 12 weeks of lactation infants are at risk from other group members and that mothers use aggression as well as restraining to protect them. Maternal aggression was negatively correlated with infant restraining. High-ranking mothers reacted aggressively to individuals handling their infants more than did middle- and low-ranking mothers. Conversely, middle- and low-ranking mothers restrained their infants more than did high-ranking mothers. Maternal aggression did not vary with infant age. Maternal aggression was directed toward a higher proportion of higher-ranking adult females and their immature offspring and was more likely to be followed by counter-aggression than nonmaternal aggression, i.e. aggression not related to interactions involving the infant. Middle-and low-ranking mothers suffered higher costs in terms of retaliation than high-ranking mothers. It is argued that the occurrence and distribution of maternal aggression among species and individuals should depend on the risk posed to infants by conspecifics as well as on the characteristics of the social structure (e.g. degree of asymmetry of agonistic contests) and of the mother (e.g. her dominance rank) which may affect the probability of retaliation.