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.     

The spatial arrangement of germ line cells in ovarian follicles of the mutantdicephalic inDrosophila melanogaster

Summary The pattern of intercellular connections between germ line cells has been studied in follicles of the mutantdicephalic (dic), which possess nurse cell clusters at both poles. Staining of follicles with a fluorescent rhodamine conjugate of phalloidin reveals ring canals and cell membranes and thus allows us to reconstruct the spatial organization of the follicle. Each germ line cell can be identified by the pattern of cell-cell connections which reflect the mitotic history of individual cells in the 16-cell cluster. The results indicate that in both wild-type anddicephalic cystocyte clusters one of the two cells with four ring canals normally becomes the pro-oocyte. However, in some follicles (dicephalic and wild-type) oocytes were found with fewer or more than four ring canals. Indic follicles, one or several nurse cells may become disconnected from the other cells during oocyte growth at stage 9–10. Such disconnected cells cannot later on empty their cytoplasm into the oocyte. This, in turn, might be of consequence for the determination of axial polarity of the embryo.     

Cat satellite DNA. Isolation using netropsin with CsCl gradients

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.     

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.     

Influence of exogenous NO donator and variations in the Ca<Superscript>2+</Superscript> content on transport activity related to tonoplast proton pumps in ontogenesis and under hyperosmotic stress

The changes in transport activity of tonoplast proton pumps under the influence of exogenous NO donator and modulation of Ca²⁺ concentration jointly and separately were investigated at different stages of ontogenesis and under hyperosmotic stress. The results suggest that both exogenous NO donator and Ca²⁺ ions can influence the activity of transport processes related to tonoplast and this influence is especially evident in the period of growth and accumulation of metabolites. Under hyperosmotic stress, H⁺-pyrophosphatase plays a more important role than H⁺-ATPase: the activity of the former increases 2.3-fold compared to the control osmotic conditions, whereas the activity of H⁺-ATPase is practically unchanged. H⁺-pyrophosphatase was more responsive to the presence of exogenous NO donator and to variations in Ca²⁺ concentration. The effects of exogenous NO donator on tonoplast proton pumps depended on the concentration of Ca²⁺, which apparently can mediate NO action.     

Structure–antimicrobial activity of some natural isocoumarins and their analogues

Three naturally occurring isocoumarins (paepalantine, paepalantine 9-O-beta-D-glucopyranoside and paepalantine 9-O-beta-D-allopyranosyl(1 --> 6) glucopyranoside) and two semi-synthetic analogues, 9,10-acylated compound and 9-OH-10-methylated compound, structurally similar to paepalantine, were evaluated for antimicrobial activity using a spectrophotometric microdilution technique. The paepalantine was active against S. aureus, S. epidermidis, and E. faecalis while the other four compounds proved ineffective against all microorganisms tested at concentrations of 500 microg/ml. Variations in phenolic substitution at OH-9 and/or OH-10 in the paepalantine molecule resulted in compounds without antimicrobial activity. A combination of structural features, two phenolic groups as cathecolic system, forms an oxygenated system arrangement that may reflect the potentially antimicrobial properties of paepalantine.     

Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.     

Stream drift,size-specific predation,and the evolution of ovum size in an amphibian

Summary A stream-breeding race of small-mouthed salamanders (Ambystoma texanum) in central Kentucky produces ova that are twice as large as those of a pond-breeding race found nearby. Embryos of stream-breeders also hatch at a more advanced developmental stage than those of pond-breeders. Morphological evidence indicates that stream-breeders were derived from pond-breeding stock. Assuming that differences between stream and pond-breeders reflect evolutionary change, and that the ancestral pond stock that invaded streams was similar to extant pond-breeders, we examined three hypotheses that might explain changes in ovum size and stage at hatching following the invasion of streams. (1) Larger ovum size evolved indirectly as a consequence of selection for rapid development which minimizes mortality risk from stream drying. (2) Increased ovum (hatchling) size and stage at hatching of stream-breeders are adaptations to resist stream current. (3) Increased ovum (hatchling) size and stage at hatching are adaptations to reduce predation on hatchlings from stream invertebrates. The results of field and laboratory studies only support hypotheses (2) and (3). Hatchlings that were relatively large or at a more advanced developmental stage had slower drift rates and were less vulnerable to predation by Phagocata gracilis, a flatworm abundant in streams in central Kentucky. Developmental and growth parameters were not correlated significantly with ovum size in populations of either geographic race. Differences in degree of parental care among races also cannot explain variation in ovum size since both races abandon their eggs immediately after oviposition.