Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

Electrophoretic evaluation of the taxonomic status of two species of sea anemone

Puerto Rican populations of two species of sea anemones (Bunodosoma cavernata and B. granulifera) which had previously been considered one were assayed electrophoretically for enzymes encoded by 12 loci. The two species shared no common allozymes at 6 of the 12 loci. Genetic distance and identity values based on these allozymes were computed for the Puerto Rican populations and for B. cavernata from Florida and B. granulifera from Panama. The Puerto Rican populations of both species had much higher genetic identities for their geographically distant conspecifics than for each other. These results indicate that the two species are reproductively isolated and should be considered as separate valid species. Average heterozygosities are presented which are the first published for coelenterate species.     

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.     

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.     

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.