Micropropagation of Rollinia mucosa (JACQ.) baill

Summary A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l⁻¹ polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l⁻¹ sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.     

Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.     

Effect of chronic ingestion of iodide during pregnancy and lactation on rat pup brain enzymes

The developmental pattern of several key enzymes in brain of pups born to mothers receiving high levels of iodide (1.1 mg daily intake) during pregnancy and lactation were followed up to the weaning period. We found that in the initial states of postnatal development, glutamic dehydrogenase increased above control levels, whereas succinic dehydrogenase decreased. At late stages, we observed differences in phosphofructokinase and malic enzyme activities which were all increased at 30 days. There was no change in hexokinase. Animal weight did not vary with respect to controls and we only obtained discrete increases (not statistically different) in serum thyroxine values, which led us to assume that the enzymatic modifications might be a consequence of either a very mild hormonal alteration or to the direct effect of iodide.     

G-11 staining in Turner's syndrome with mos 45,X/46,X,r(?)

Mos 45,X/46,X,r(?) in 4 patients with Turner's syndrome and no signs of virilization, and in one pair of monozygotic twins, one of them with clitoral hypertrophy, was studied using combined cytogenetic techniques and specially G-11 staining for the characterization of the X or Y origin of the rings. In all 6 patients the ring was G-11 positive, attesting its Y origin. Both twins were operated and bilateral streak gonads with a bilateral nodule of testicular tissue were found. Similar small rings were also studied in one patient with mos 46,XX/46,X,r(X) and in one nonvirilized Turner's syndrome patient with a larger ring; in these two cases the ring was G-11 negative. It seems that the small rings occasionally found in Turner's syndrome are more frequently from Y origin and therefore prophylactic gonadectomy should be considered.     

Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies

Protein G is an immunoglobulin (IgG)-binding bacterial cell wall protein recently isolated from group G streptococci. We have investigated the avidity of protein G for various monoclonal and polyclonal Ig of the IgG class, and compared it with the binding properties of protein A, the staphylococcal Fc-binding protein. Radiolabeled Ig were mixed with Sepharose-coupled protein G or protein A, and the amounts of radioactivity bound to the matrix-coupled bacterial proteins were determined. The avidity was found to be greater for protein G than for protein A for all examined Ig. Protein G bound all tested monoclonal IgG from mouse IgG1, IgG2a, and IgG3, and rat IgG2a, IgG2b, and IgG2c. In addition, polyclonal IgG from man, cow, rabbit, goat, rat, and mouse bound to protein G, whereas chicken IgG did not. The binding property of protein G was additionally exploited in the Western blot assay, in which iodine-labeled protein G was used successfully for the detection of a rat monoclonal antibody against ovalbumin, and for the detection of rabbit and goat polyclonal whole antisera against human urinary proteins. In these experimental situations, protein G was found to be a powerful reagent for the detection of IgG, and consequently the antigen against which these antibodies are directed.     

Chromosomal location of the active NORs in theSteropleurus martorelli complex

The chromosomal location of the active NORs has been analyzed by a silver impregnation procedure in theSteropleurus martorelli complex. A primary NOR, which is always present at the first meiotic prophase, has been found in each of the four described races. In addition to this, all races possess one or two secondary NORs which are less active than the former and can be occasionally shown. Usually only one of the two homologous chromosomes has been found to be involved with nucleolus organisation.These results are discussed in relation to hypotheses on the chromosome differentiation of this species complex.