Vibrio parahaemolyticus thermostable direct hemolysin can induce an apoptotic cell death in Rat-1 cells from inside and outside of the cells

Rat-1 cells exposed to Vibrio parahaemolyticus thermostable direct hemolysin (TDH) developed morphological changes including shrinkage of the cells and reduction in the size of nuclei. Cells either microinjected with TDH or transfected with the tdh gene also showed morphological changes similar to those induced by externally added toxin. Furthermore, TDH-exposed or tdh-transfected cells both showed chromatin condensation and DNA fragmentation which suggest cells undergoing apoptosis. In contrast, expression of a TDH mutant (R7) did not reveal any cytotoxic effects. We demonstrate that expressed TDH was distributed in the cytoplasm. The interleukin-1beta-converting enzyme-related protease inhibitor ZVAD-FMK did not inhibit TDH cytotoxicity. Our results suggest that TDH can induce its cytotoxicity both from outside and from inside the cells and killed the cells through apoptosis.     

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.     

Secretion of bovine alpha-lactalbumin into the milk of transgenic rats.

Bovine alpha-lactalbumin (b alpha LA) gene prepared by polymerase chain reaction was introduced into rat genomes by microinjection. Out of 17 transgenic rat lines, 11 secreted b alpha LA into their milk at concentrations higher than 0.2 micrograms/ml. Of these, three lines secreted b alpha LA at concentrations higher than those in bovine milk. The highest concentration of b alpha LA secreted into rat milk was 2,400 micrograms/ml.     

Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

Endothelin-1 exerts its actions via activation of ET_A and ET_B G_q/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET_B receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca²⁺ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET_B-transfected cells and endothelial cells; this response is sensitive to ET_B but not ET_A receptor blockade. In endothelial cells, the endothelin-1-induced Ca²⁺ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET_B-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET_B receptors and increase cytosolic Ca²⁺ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET_B to induce nitric oxide formation, thus modulating endothelial function.     

BCAR3 regulates EGF-induced DNA synthesis in normal human breast MCF-12A cells

BCAR3 (breast cancer anti-estrogen resistance 3) is a signal transducer containing an SH2 domain, a proline/serine-rich domain and a GDP-exchange factor homologous domain, whose role in signaling pathways is currently unclear. Furthermore, BCAR3 is implicated in anti-estrogen resistance of breast cancer cells. In the present study, we investigated the functional role of BCAR3 in a mitogenic signaling pathway of EGF in non-tumorigenic human breast epithelial MCF-12A cells. Microinjection of an anti-BCAR3 antibody, siRNAs targeting BCAR3 and an SH2 domain of BCAR3 inhibited EGF-induced DNA synthesis. Direct association of BCAR3 with activated EGF receptor and Cas was observed. Lastly, microinjection of a BCAR3 expression plasmid induced DNA synthesis. These findings suggest that the BCAR3 protein, through its SH2 domain, is involved in the signaling pathways of EGF leading to cell cycle progression, and that BCAR3 itself is part of a mitogenic signaling pathway.     

Rearrangements of microinjected recombinant DNA in the genome of transgenic mice

Summary Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic F_o mouse by the plasmid rescue technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.     

Caffeine inhibition of cytokinesis: effect on the phragmoplast cytoskeleton in livingTradescantia stamen hair cells

Summary The distribution of microtubules and actin microfilaments during caffeine-induced inhibition of cell plate formation has been studied in livingTradescantia stamen hair cells. Previous studies have shown that caffeine allows cell plate initiation but prevents its completion, resulting in binucleate cells. In the present study, confocal microscopy of cells microinjected with fluorescent brain tubulin or phalloidin, and cultured in the presence 5 mM caffeine, revealed that the initiation and early lateral expansion phase of the phragmoplast occur normally. However, caffeine completely inhibits the formation of the cytoskeletal torus which occurs in untreated cells during the late stages of cell plate and phragmoplast expansion. Caffeine further causes the disintegration of the incomplete cell plate. The results allow us to distinguish two phases in cell plate and phragmoplast growth: the initiation and early expansion phase, which is not affected by caffeine, and the late lateral expansion phase, which is completely inhibited in the presence of caffeine. Also in this study, the use of a high phalloidin concentration has revealed structural detail about the actin microfilaments involved in cell plate formation: microfilaments are observed that link the expanding edge of the phragmoplast with the cortical division site. In addition, cortical actin patches are observed within the actin depleted zone that might play a role in guidance of phragmoplast and cell plate expansion.     

Intracellular polycationic molecules cause reversible swelling of the rough endoplasmic reticulum

The microinjection of polycationic but not anionic molecules causes swelling of the rough endoplasmic reticulum (RER) in salivary gland cells of a fly larva. Ca-EGTA buffers, lanthanum chloride, lysozyme, bovine serum albumin, cationic and anionic ferritin were microinjected into salivary gland cells and their effects observed by light and electron microscopy. Immediately after the microinjection of polycationic molecules, the cytoplasm changed from transparent to opaque as the RER became swollen. Binding of polycationic molecules to the RER may cause the membrane to become permeable to some solute and swell due to osmotic forces.