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.     

Separation of plant membranes by electromigration techniques

The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Transport of GABA at the Blood-CSF Interface

Abstract: The entry of GABA into cerebrospinal fluid (CSF) was studied in dogs anesthetized with pentobarbital and relaxed with suxamethonium. GABA was administered intravenously as a priming dose and subsequent maintenance infusion to compensate for the rapid elimination of the amino acid. Steady state concentrations of GABA in CSF were reached between 10 and 60 min after injection, the rate of entry tending to decrease with increasing plasma levels. During steady state conditions CSF concentrations showed great interin-dividual differences and varied between 0.03 and 5.1% of those in plasma. Probenecid and sodium valproate considerably enhanced the CSF/plasma concentration ratio of GABA. When GABA was directly injected into the liquor space, probenecid slowed down the elimination of GABA from CSF. The results suggest a transport of GABA into and out of CSF, the outward transport being inhibited by probenecid and sodium valproate.     

Parasite-induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster

Larval hemolymph tyrosinase activity in Drosophila melanogaster was detected with high performance liquid chromatography with electrochemical detection. The enzyme hydroxylated L-tyrosine, and oxidized the diphenol substrates L-dopa and dopamine. In larvae of a selected immune-reactive strain the rates of tyrosine hydroxylation, dopa oxidation, and dopamine oxidation were markedly increased during the early stages of melanotic encapsulation of the eggs of the parasitic wasp Leptopilina boulardi. Tyrosinase activity was not modified in parasitized larvae of a selected susceptible strain of D. melanogaster, in which hosts the parasitoids developed unmolested. During the same period of parasitization, the amount of free tyrosine in immune reactive larvae was approximately three times higher than in susceptible hosts. These data indicate that the tyrosinase system of the immune reactive strain is activated during parasitization, and this results in the synthesis of some precursors which ultimately produce a melanotic and sclerotic capsule around the eggs of the parasite. Based on known genetic information of the enzyme system in Drosophila, it appears that at least two genes may be involved in the activation process, one associated with the proenzyme for monophenol oxidase activity, and the second with the proenzyme for diphenol oxidase activity.     

On the structure and chromosome location of the 72- and 92-kDa human type IV collagenase genes.

The 72- and 92-kDa type IV collagenases are members of a group of secreted zinc metalloproteases. Two members of this family, collagenase and stromelysin, have previously been localized to the long arm of chromosome 11. Here we assign both of the two type IV collagenase genes to human chromosome 16. By sequencing, the 72-kDa gene is shown to consist of 13 exons, 3 more than have been reported for the other members of this gene family. The extra exons encode the amino acids of the fibronectin-like domain which has so far been found in only the 72- and 92-kDa type IV collagenase. The evolutionary relationship among the members of this gene family is discussed.     

Cell adhesion molecule in the hexactinellid Aphrocallistes vastus

Abstract. The Hexactinellida sponge Aphrocallistes vastus contains a soluble aggregation factor (AF) whose purification has been described in this communication. It is characterized by a S°_20.w value of 37 and a buoyant density of 1.45 g/cm³. The AF is a glycoporteinaceous particle composed of three major protein species; no core structure could be visualized. In the presence of Ca²⁺, the AF causes secondary aggregation of single cells. The aggregation process is temperature, pH, and ionic strength independent within a broad range. Evidence is presented indicating that two (or more) AF molecules are required for the establishment of a stable cell: cell interaction. In contrast to the AFs from demosponges, the hexactinellid AF functions species-unspecifically.