Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

Segments of the POU domain influence one another''s DNA-binding specificity.

The ubiquitously expressed mammalian POU-domain protein Oct-1 specifically recognizes two classes of cis-acting regulatory elements that bear little sequence similarity, the octamer motif ATGCAAAT and the TAATGARAT motif. The related pituitary-specific POU protein Pit-1 also recognizes these two motifs but, unlike Oct-1, binds preferentially to the TAATGARAT motif. Yet in our assay, Pit-1 still binds octamer elements better than does the octamer motif-binding protein Oct-3. The POU domain is responsible for recognizing these diverse regulatory sequences through multiple DNA contacts that include the two POU subdomains, the POU-specific region, and the POU homeodomain. The DNA-binding properties of 10 chimeric POU domains, in which different POU-domain segments are derived from either Oct-1 or Pit-1, reveal a high degree of structural plasticity; these hybrid proteins all bind DNA well and frequently bind particular sites better than does either of the parental POU domains. In these chimeric POU domains, the POU-specific A and B boxes and the hypervariable POU linker can influence DNA-binding specificity. The surprising result is that the influence a particular segment has on DNA-binding specificity can be greatly affected by the origin of other segments of the POU domain and the sequence of the binding site. Thus, the broad but selective DNA-binding specificity of Oct-1 is conferred both by multiple DNA contacts and by dynamic interactions within the DNA-bound POU domain.     

Isolation and partial characterization of a protein kinase NII from wheat germ chromatin

A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (-³²P)ATP and (-³²P)GTP.     

Spermatophore development and sperm ultrastructure inCraterostigmus tasmanianus (Chilopoda,Craterostigmomorpha)

 Spermatophore development and ultrastructure of the mature sperm of Craterostigmus tasmanianus were studied using light and electron microscopy. In C. tasmanianus, as in the Scolopendromorpha, the spermatophore develops within the vas deferens. The latter consists of three parts, each with a different morphology. The first may be involved in guiding the sperm to roll up into typical ring-like structures, while the other two, which show an evident secretory activity, secrete the acellular wall of the spermatophores. The ultrastructure of mature spermatozoa showed that a very close similarity exists between Craterostigmomorpha and Lithobiomorpha, especially regarding the organization of the connecting piece. Based on this similarity, we consider the Craterostigmomorpha together with the Scolopendromorpha, Geophilomorpha and Lithobiomorpha (=Pleurostigmophora) to be the sister group of the Scutigeromorpha. Accepted: 2 June 1996     

Sex chromosome polymorphism and heterogametic males revealed by two cloned DNA probes in the ZW/ZZ fish Leporinus elongatus

In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA of males and females of the rainbow trout (XX/XY) and of Leporinus elongatus (ZW/ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinus friderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.     

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.     

Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco-protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against 7. cruzi.