Excitatory Amino Acid Receptors Coupled to the Phosphoinositide Pathway in Bergmann Glia

Glutamate (L-glu) receptors coupled to phosphoinositide hydrolysis in primary cultures of Bergmann cells from chick cerebellum were characterized biochemically and pharmacologically. Both ionotropic and metabotropic receptor agonists stimulated [³H] inositol phosphates accumulation in the following order of potency: QA>NMDA>L-glu>KAQA>AMPA>>t-ACPD. QA showed a biphasic dose-response curve (EC₅₀ = 0.07 and 53 M), suggesting interaction with two populations of receptors; L-glu was the most efficient agonist. Stimulation by NMDA was blocked by CPP, APS and MK-801; that by AMP A and KA was inhibited 100% by CNQX and DNQX, whereas the effect of QA was decreased both by CNQX and the metabotropic antagonist 4-CPG. Stimulation of PIP₂ hydrolysis induced by metabotropic L-glu receptor agonist t-ACPD was blocked by 4-CPG but was only moderately inhibited by MCPG. EAA-induced [³H]IPs accumulation was dependent on external Ca²⁺ and was not affected by nifedipine verapamil, or dantrolene; thapsigargin increased the effect. Results suggest that EAA activate the PI pathway in Bergmann glia through ionotropic (NMDA and AMPA/KA) as well as metabotropic receptor subtypes (t-ACPD) which could act jointly influencing neurotransmission at the parallel fiber—Purkinje cell synapses in the cerebellum.     

Molecular Cloning of α-Amylases from Cotton Boll Weevil, Anthonomus grandis and Structural Relations to Plant Inhibitors: An Approach to Insect Resistance

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of α-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two α-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5′ and 3′ RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7 kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several α-amylase inhibitors from plants were assayed against A. grandis α-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and α-AI1 inhibitors on A. grandis α-amylase activity. This work suggests that genetic engineering of cotton to express α-amylase inhibitors may offer a novel route to A. grandis resistance.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

PSSA-2, a Membrane-Spanning Phosphoprotein of Trypanosoma brucei,Is Required for Efficient Maturation of Infection

The coat of Trypanosoma brucei consists mainly of glycosylphosphatidylinositol-anchored proteins that are present in several million copies and are characteristic of defined stages of the life cycle. While these major components of the coats of bloodstream forms and procyclic (insect midgut) forms are well characterised, very little is known about less abundant stage-regulated surface proteins and their roles in infection and transmission. By creating epitope-tagged versions of procyclic-specific surface antigen 2 (PSSA-2) we demonstrated that it is a membrane-spanning protein that is expressed by several different life cycle stages in tsetse flies, but not by parasites in the mammalian bloodstream. In common with other membrane-spanning proteins in T. brucei, PSSA-2 requires its cytoplasmic domain in order to exit the endoplasmic reticulum. Correct localisation of PSSA-2 requires phosphorylation of a cytoplasmic threonine residue (T₃₀₅), a modification that depends on the presence of TbMAPK4. Mutation of T₃₀₅ to alanine (T₃₀₅A) has no effect on the localisation of the protein in cells that express wild type PSSA-2. In contrast, this protein is largely intracellular when expressed in a null mutant background. A variant with a T₃₀₅D mutation gives strong surface expression in both the wild type and null mutant, but slows growth of the cells, suggesting that it may function as a dominant negative mutant. The PSSA-2 null mutant exhibits no perceptible phenotype in culture and is fully competent at establishing midgut infections in tsetse, but is defective in colonising the salivary glands and the production of infectious metacyclic forms. Given the protein''s structure and the effects of mutation of T₃₀₅ on proliferation and localisation, we postulate that PSSA-2 might sense and transmit signals that contribute to the parasite''s decision to divide, differentiate or migrate.     

TNF-α Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

Background

Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS.

Principal Findings

We show that TNF-α is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression.

Conclusion

Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.     

Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.     

Mesozooplankton of the Kosi Bay lakes,South Africa

The Kosi coastal lake system, a chain of four interconnected basins, is located in the subtropical north-eastern corner of South Africa. Little information is available on zooplankton of the system and the main aim of this study is to report on zooplankton samples collected during 2002 and 2003. The set of samples consists of seasonal, subsurface mesozooplankton samples that were collected during nighttime in each of the lakes. A well-developed salinity gradient was evident along the interconnected lakes in the subsurface water during all seasons, ranging from freshwater in the upper lake Amanzamnyama to a maximum of 22 recorded in Lake Makhawulani. The zooplankton community structures of the lakes reflected the salinity gradient of the system, with some coastal marine taxa recorded in the lakes closer to the mouth and only freshwater taxa recorded in Lake Amanzamnyama. Mesozooplankton diversity and abundance were relatively low compared to other estuarine systems along the eastern coast of South Africa. The dominant taxa were calanoid copepods Acartiella natalensis and Pseudodiaptomus stuhlmanni and the mysid Mesopodopsis africana in the lower lakes, whereas cyclopoids Mesocyclops sp. and Thermocyclops sp. dominated the freshwater lake Amanzamnyama.     

<Emphasis Type="Italic">Tumor necrosis factor</Emphasis> (<Emphasis Type="Italic">TNF</Emphasis>) and <Emphasis Type="Italic">TNFR1</Emphasis> polymorphisms are not risk factors for rheumatoid arthritis in a Mexican population

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of rheumatoid arthritis (RA). Different genetic variants including the TNF ?308G/A polymorphism are associated with RA susceptibility. However, these findings have not been replicated in all populations. The aim of this study was to determine whether the TNF ?1031T/C (rs1799964), ?376G/A (rs1800750), ?308G/A (rs1800629) ?238G/A (rs361525), and TNFR1 ?609G/T polymorphisms are associated with RA susceptibility in a sample of Mexican patients. Our study included 499 patients with RA and 492 healthy controls. The genotypes of the TNF polymorphisms were obtained using TaqMan assay. The genotype and allele frequencies of the TNF ?1031T/C, ?376G/A, ?308G/A, ?238G/A, and TNFR1 ?609G/T polymorphisms were similar among RA cases versus healthy controls, and no association with RA susceptibility was identified. Our results suggest that the TNF ?1031T/C, ?376G/A, ?308G/A, ?238G/A, and TNFR1 ?609G/T polymorphisms are not associated with RA susceptibility in a sample of Mexican patients.