In Vivo RNA Visualization in Plants Using MS2 Tagging.

Intracellular trafficking and asymmetric localization of RNA molecules within cells are a prevalent process across phyla involved in developmental control and signaling and thus in the determination of cell fate. In addition to intracellular localization, plants support the trafficking of RNA molecules also between cells through plasmodesmata (PD), which has important roles in the cell-to-cell and systemic communication during plant growth and development. Viruses have developed strategies to exploit the underlying plant RNA transport mechanisms for the cell-to-cell and systemic dissemination of infection. In vivo RNA visualization methods have revolutionized the study of RNA dynamics in living cells. However, their application in plants is still in its infancy. To gain insights into the RNA transport mechanisms in plants, we study the localization and transport of Tobacco mosaic virus RNA using MS2 tagging. This technique involves the tagging of the RNA of interest with repeats of an RNA stem-loop (SL) that is derived from the origin of assembly of the bacteriophage MS2 and recruits the MS2 coat protein (MCP). Thus, expression of MCP fused to a fluorescent marker allows the specific visualization of the SL-carrying RNA. Here we describe a detailed protocol for Agrobacterium tumefaciens-mediated transient expression and in vivo visualization of MS2-tagged mRNAs in Nicotiana benthamiana leaves.

Cr(VI) and COD removal from landfill leachate by polyculture constructed wetland at a pilot scale.

Four subsurface horizontal-flow constructed wetlands (CWs) at a pilot scale planted with a polyculture of the tropical plants Gynerium sagittatum (Gs), Colocasia esculenta (Ce) and Heliconia psittacorum (He) were evaluated for 7 months. The CW cells with an area of 17.94 m(2) and 0.60 m (h) each and 0.5 m of gravel were operated at continuous gravity flow (Q = 0.5 m(3) day(-1)) and a theoretical HRT of 7 days each and treating landfill leachate for the removal of filtered chemical oxygen demand (CODf), BOD5, TKN, NH4 (+), NO3 (-), PO4 (3-)-P and Cr(VI). Three CWs were divided into three sections, and each section (5.98 m(2)) was seeded with 36 cuttings of each species (plant density of six cuttings per square metre). The other unit was planted randomly. The final distributions of plants in the bioreactors were as follows: CW I (He-Ce-Gs), CW II (randomly), CW III (Ce-Gs-He) and CW IV (Gs-He-Ce). The units received effluent from a high-rate anaerobic pond (BLAAT®). The results show a slightly alkaline and anoxic environment in the solid-liquid matrix (pH = 8.0; 0.5-2 mg L(-1) dissolved oxygen (DO)). CODf removal was 67 %, BOD5 80 %, and TKN and NH4 (+) 50-57 %; NO3 (-) effluents were slightly higher than the influent, PO4 (3-)-P (38 %) and Cr(VI) between 50 and 58 %. CW IV gave the best performance, indicating that plant distribution may affect the removal capacity of the bioreactors. He and Gs were the plants exhibiting a translocation factor (TF) of Cr(VI) >1. The evaluated plants demonstrated their suitability for phytoremediation of landfill leachate, and all of them can be categorized as Cr(VI) accumulators. The CWs also showed that they could be a low-cost operation as a secondary system for treatment of intermediated landfill leachate (LL).

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine.

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20-28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application.

Onchocerca volvulus: immunohistochemical and immunoelectron microscopical distribution of a polyamine oxidizing enzyme.

We studied the distribution of a polyamine oxidizing enzyme (PAO) in Onchocerca volvulus and other nematode parasites by immunohistochemistry and electron microscopy with immunogold technique using a polyclonal antiserum raised against purified PAO from Ascaris suum. In adult O. volvulus the protein was localized in the outer zone and the area of the basal labyrinth of the hypodermis and occasionally in the outer zone of the uterine epithelium. Further, the fluid in the body cavity was strongly stained. No specific labelling was observed in the cuticle, muscles, epithelia of intestine, ovaries, testis and vas deferens or in sperm, oocytes and embryos. Third-stage larvae of O. volvulus in Simulium soubrense showed strong staining; the same was observed in Anisakis sp. larvae, where the inner and outer zone of the hypodermis were strongly labelled. All mature, intact and dead microfilariae in nodules, skin and lymph nodes were well stained and it was possible to show that the cytoplasm of the hypodermal cells, but not the mitochondria, nuclei or other organelles of muscle cells, was preferentially labelled by immunogold particles. Investigation of adult A. suum presented specific labelling of the hypodermis, but the basal labyrinth was more strongly marked than the outer zone.

Cellular and subcellular localization of cationic leukocyte antigen (CLA) in human eosinophil granulocytes.

The cellular and subcellular localization of cationic leukocyte antigen (CLA) in human peripheral blood and tissue granulocytes was investigated by immunoenzymatic labeling and by immunoelectron microscopy. Human peripheral blood granulocytes from healthy individuals and from subjects with eosinophilia of varying etiology, as well as intravascular and/or perivascular granulocytes in skin biopsies taken from patients with generalized onchocerciasis, a skin disease caused by microfilariae of Onchocerca volvulus, were studied. Controlled indirect immunoenzymatic staining for CLA in cytospin preparations of buffy coat cells and in histological sections of skin biopsies revealed that this protein was exclusively found in the cytoplasm of eosinophil granulocytes. Furthermore, immunogold labeling coupled with electron microscopy showed that CLA was specifically localized within the matrix of both the small non-crystalloid-containing pale granules and the large crystalloid-containing secondary granules of peripheral blood and tissue eosinophils. No specific gold labeling was observed in other organelles of eosinophils, in neutrophils, basophils, lymphocytes or monocytes.

Neutrophil granule proteins: evidence for the participation in the host reaction to skin microfilariae of Onchocerca volvulus after diethylcarbamazine administration.

The participation of neutrophil granulocytes in the cellular reaction to skin microfilariae of Onchocerca volvulus was studied by immunohistochemistry. Skin biopsies were obtained from adult Liberian and Ugandan patients with generalized onchocerciasis after exposure to topically applied diethylcarbamazine (DEC) and from untreated patients. After DEC many damaged microfilariae were observed either in dermal infiltrates or in epidermal microabscesses consisting both of neutrophils and eosinophils. Infiltrates and microabscesses contained some intact granulocytes and many neutrophils releasing myeloperoxidase, elastase, lactoferrin, defensin, lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. Eosinophils discharged peroxidase and cationic proteins. Released granule proteins and remnants of disrupted granulocytes were found on the surface and in close proximity of damaged microfilariae in dermal infiltrates and epidermal microabscesses. In larger microabscesses neutrophils were predominant. These observations show that neutrophils and not only eosinophils recruit, accumulate, localize around and release their helminthotoxic granule proteins such as myeloperoxidase onto or closely around skin microfilariae of O. volvulus after topical DEC administration. The association between these processes and the damage of the microfilariae indicated that neutrophils together with eosinophils attack and damage microfilariae of O. volvulus after DEC treatment in the skin.

[Cytomegalovirus infection in kidney transplantation]. / Infecção por citomegalovírus no transplante renal.

150 patients, subjected to kidney graft transplantation between January 1988 and December 1989, were studied. Mean +/- 95% CL age was 37.5 +/- 2.03 (range 12-69) years. IgG and IgM antibodies levels (ELISA) cytomegalovirus (CMV) were investigated in the donor before organ harvesting and in the kidney recipients on 1st and 21th days and then on the 3rd, 6th and 9th months after transplantation. Patients lacking either donor or 1st day studies were excluded. 133 donor (D) receptor (R) pairs were classified as group 1) D+/R+, 2) D+/R-, 3) D-/R+ e 4) D-/R-. Prevalence, severity of CMV disease and date of diagnosis were studied. Mean time +/- 95% CI of diagnosis after transplantation was 78.4 +/- 15.1 days. Seronegative receptors had a statistically significant higher prevalence of the disease as to seropositive receptors, but not a higher incidence of disease severity. One out of five patients had a serious form of disease. Hiperimmune globulin was used in 15 patients (all serious forms and 3 moderate forms of of disease). No patient died as a result of CMV infection.

Scanning electron microscopic study of adults and microfilariae of Dunnifilaria meningica (Filarioidea: Onchocercidae).

Dunnifilaria meningica from naturally infected Neotoma micropus in Mexico were studied by scanning electron microscopy (SEM). The anterior end of adults is surrounded by two pairs of buccal and two pairs of cervical papillae. Two amphidial openings lie near the central mouth opening, surrounded by a thick cuticular ring. The cuticular surface of males and females shows fine transversal striations. At the posterior end of the male is a semicircular cloaca, a large preanal central papilla, two pairs of perianal and two pairs of postanal papillae. The short, strong, and almost equal spicules are rolled plates that end in a knob-shaped apex, showing a central groove. In the female, the vulva is located 550 microns from the anterior end. The inconspicuous anus is subterminally situated in the right ventrolateral portion of the posterior end. The anterior tip of the sheathed microfilariae is formed by a cap-like disk. Cuticular annulations were clearly demonstrated across the body.

Dunnifilaria meningica sp. n. (Filarioidea: Onchocercidae) from the central nervous system of the wood-rat (Neotoma micropus) in Mexico.

Dunnifilaria meningica sp. n. (Filarioidea: Onchocercidae) is described from the wood-rat, Neotoma micropus, trapped in Nuevo Leon State, Mexico. The adult worms are characteristically found in the subarachnoid spaces along the cerebellum and the medulla oblongata. The short, sheathed microfilariae are found in the peripheral blood. They do not show a periodicity. The adults are of small size (female worms are approximately 50 mm long, males about 25 mm). Female worms are didelphic. Male worms have subequal, dissimilar spicules and peri- and postanal papillae. This is the third species of the genus Dunnifilaria. It differs from the other species in its habitat in the host, the size of the body, the female tail length, the number and arrangement of the caudal papillae.