The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.     

Mechanism of action of ryanodine on cardiac sarcoplasmic reticulum

Ryanodine was found to initially inhibit calcium uptake by cardiac sarcoplasmic reticulum. This initial depression was followed by a later marked stimulation of calcium uptake. These effects were noted when calcium uptake was measured in the presence or absence of oxalate. The requirement for preincubation with ryanodine was highly dependent on ryanodine concentration and temperature. The mechanism of action of ryanodine clearly was not an effect on oxalate entry or calcium oxalate precipitation because the effects were also observed in the absence of oxalate. Ryanodine also had no effect on passive calcium efflux from actively loaded vesicles. Because ryanodine had no effect on Ca2+-ATPase activity under defined conditions of an ATP-regenerating system and no calcium gradient, we suggest ryanodine does not change the stoichiometry of the pump. Our results are consistent with the hypothesis that ryanodine closes a calcium channel in a subpopulation of the vesicles.     

β-Xylosidases from filamentous fungi: an overview

β-Xylosidases are hydrolytic enzymes which play an important role in xylan degradation, hydrolyzing xylobiose and xylooligosaccharides to xylose from the non-reducing end. Filamentous fungi are particularly interesting producers of this enzyme from an industrial point of view, due to the fact that they secrete β-xylosidases into the medium. Besides, fungal β-xylosidases are highly advantageous for their elevated activity levels and specificity. Interest in xylanolytic enzymes has been increasing, for their possible application in many biotechnological processes. This fact has driven the isolation, purification and characterization of several β-xylosidases. In this review, the mechanisms of action, substrate specificities, physicochemical characteristics, regulation at molecular level, molecular cloning and classification of filamentous fungal β-xylosidases are described. The potential industrial applications of fungal β-xylosidases will also be presented.     

The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi . Therefore the gene sequence S - Phi - Hal -H- Po2 -Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.     

Content of red blood-cell sialic acid in BW 35 blood donors. Relation to magnesium concentration and pyruvate kinase activity

Previous studies have shown that the concentration of red blood cell (RBC) magnesium is significantly lower in subjects carrying an HLA-BW 35 antigen (p less than 0.001) than in non-carriers. As this finding might be related to modifications of the RBC membrane sialoglycoconjugates, RBC sialic acid was comparatively determined in BW 35+ and BW 35- subjects. Pyruvate-kinase activity mean RBC volume, and reticulocyte count have also been determined in order to estimate whether some significant variations in the level of these age markers could be detected between the HLA BW 35+ and BW 35- subjects. A significant negative correlation between sialic acid and RBC magnesium concentrations was observed for the whole population tested (n 57, p less than 0.005), 61% of the BW 35+ and only 25% of the BW 35- individuals having sialic acid values above, and magnesium values below the overall mean (p less than 0.01). The variance of mean RBC volume was also larger for the BW 35+ group. Other determinations did not show any significant variations, suggesting that the results are not related to RBC age.     

Protective reaction of the myocardium in poisoning

Intensive synthesis of collagen-like substance was revealed in the rabbit myocardium during experimental diphtheria intoxication. It was more marked in the right ventricle 24 hours after the injection of diphtheria toxin. Since similar changes (the substance was mainly formed around blood vessels) have been observed in other cases of toxic myocardial alterations (i.e. ethanol intoxication, injection of pharmacological agents, etc.), it can be assumed that it is a standard protective reaction of the altered heart to the penetration of toxic agents from the blood into the myocardial tissue.     

Indirect assays for deoxyhypusine hydroxylase using dual-label ratio changes and oxidative release of radioactivity

Two procedures for rapid assay of deoxyhypusine hydroxylase activity are described. One of these assays measures changes in the 3H:14C ratio of dual-labeled protein that results from the release of tritium from a specific position in the side chain of the 3H,14C-labeled constituent amino acid deoxyhypusine upon its conversion to [3H,14C]hypusine. The other assay relies upon release of radioactivity from product protein by periodate oxidation of the radiolabeled side chain of component hypusine. The good correspondence of each of these assays with the ion exchange chromatographic method which measures hypusine and deoxyhypusine in acid hydrolysates of protein indicates that each provides a valid means of determining deoxyhypusine hydroxylase activity.     

Effects of EEG of the osmotic opening of the blood-brain barrier in rats

EEG activity was recorded in rats submitted to osmotic opening of the BBB by intracarotid mannitol infusion.This procedure produced an immediate short-lasting depression of the EEG and a tardive paroxysmal EEG activity. Both these phenomena were more relevant on the ipsilateral hemisphere. In some instances a tonico-clonic seizure was recorded.Pre-treatment with diazepam abolished the occurrence of the tardive EEG and behavioral modifications.In accord with previous findings, focal seizure activity is likely to be responsible for the metabolic abnormalities associated with osmotic opening of the BBB. This preparation therefore produces in the brain unphysiological states in respect to local metabolism and electrical function.