Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1

Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.     

Cloning,expression, and genomic organization of mouse mp29 gene

The convulsions of approximately 25% of epileptics are inadequately controlled by currently available medication; therefore the preparation of new antiepileptic drugs is of great interest. Aryl semicarbazones can be considered a new class of compounds presenting anticonvulsant activity. In addition, they can be orally administered and are more active as anticonvulsants than mephenytoin or phenobarbital. However, one disadvantage of these compounds is their low water solubility. As a strategy to circumvent this problem, a 1:1 inclusion compound of benzaldehyde semicarbazone (BS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was prepared and characterized. The anticonvulsant activities of the free semicarbazone and of the inclusion compound were evaluated in rats using the maximum electroshock and audiogenic seizures screenings. In both tests the minimum dose of compound necessary to produce activity decreases from 100mg/kg for the free semicarbazone to 35 mg/kg for the inclusion compound, indicating a significant increase in the bio-availability of the drug.     

The antioxidant cocktail,effective microorganism X (EM-X), protects retinal neurons in rats against N-methyl-D-aspartate excitotoxicity in vivo

Injection of the glutamate agonist N-methyl-D-aspartate (NMDA) into the vitreous body of rats resulted in severe degeneration of neurons in the retina, with a loss of 81% of ganglion cells and 43% of non-ganglion cells. The cocktail EM-X is a novel antioxidant drink derived from ferment of unpolished rice, papaya and sea-weeds with effective microorganisms (EM-X). In animals treated with an intraperitoneal injection of EM-X, the loss of ganglion cells was reduced to 55% and that of non-ganglion cells to 34% when compared to untreated NMDA-injected retinas. Cell degeneration resulting from NMDA excitotoxicity, is thought to be mediated via oxidative stress mechanisms. The neuroprotective effect of the EM-X in this system is therefore likely to be due, at least in part, to its flavonoids, saponins, vitamin E and ascorbic content.     

Evaluation of false transrectal ultrasonographic pregnancy diagnoses in sheep by measuring the plasma level of pregnancy-associated glycoproteins

The present study was undertaken to investigate to what extent pregnancy diagnoses made by transrectal ultrasonography could be confirmed by measurements of plasma concentration of ovine pregnancy-associated glycoproteins (ovPAG). A total of 424 Awassi x Merino ewes were synchronized for estrus and examined by transrectal ultrasonography. In Experiment 1, the ewes (n = 156) were repeatedly scanned in a standing position on d 29, 36 and 50 of gestation. Similarly, the ewes (n = 268) in Experiment 2 were scanned on d 24, 29 and 34 of gestation, but these ewes were fasted for 12 h prior to the examination and the abdominal wall of each animal was lifted up by the hands of the assistant during the scanning. Blood samples were withdrawn after each transrectal ultrasonographic examination in both experiments. Ovine PAG concentrations were measured in plasma by a heterologous radioimmunoassay and the cut-off value for pregnancy was > or = 1 ng.mL-1. Based on the lambing performance, in Experiment 1, altogether 47 false negative and 38 false positive diagnoses were made by transrectal ultrasonography in 24 and 33 ewes, respectively between d 29 and 50 of gestation. In Experiment 2, altogether 8 false negative and 13 false positive diagnoses both were made in 7 ewes between d 24 and 34 of gestation. In both experiments, all ewes with false negative diagnoses had ovPAG concentrations higher than the threshold level for pregnancy diagnosis and all ewes with false positive diagnoses had ovPAG concentrations lower than the threshold of pregnancy. Furthermore, by the PAG-RIA test all lambed or aborted ewes (n = 63) were correctly diagnosed as pregnant and with three exceptions, all non-lambed ewes (n = 361) were correctly diagnosed as non-pregnant during the examined periods of both experiments.     

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10.

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.