Identification of differentially expressed cDNA sequences in ovaries of sexual and apomictic plants of Brachiaria brizantha

The isolation of genes associated with apomixis would improve understanding of the molecular mechanism of this mode of reproduction in plants as well as open the possibility of transfer of apomixis to sexual plants, enabling cloning of crops through seeds. Brachiaria brizantha is a highly apomictic grass species with 274 tetraploid apomicts accessions and only one diploid sexual. In this study we have compared gene expression in ovaries at megasporogenesis and megagametogenesis of sexual and apomictic accessions of B. brizantha by differential display (DD-PCR), with 60 primer combinations. Specificity of 65 cloned fragments, checked by reverse northern blot analysis, showed that 11 clones were differentially expressed, 6 in apomictic ovaries, 2 in sexual and 3 in apomictic and sexual, but at different stages. Of the 6 sequences isolated that were preferentially expressed in the apomictic accession: one sequence was from ovaries at megasporogenesis stage; three were from megagametogenesis stage; two were from both stages. Of the two sequences isolated from the sexual accessions, one showed expression in ovaries at megagametogenesis, while the other sequence was shown to be specific to both stages. Three sequences were from megasporogenesis stage in apomicts but were also detected at megagametogenesis in sexual plants. Sequence analysis showed that 5 of the 11 clones had no apparent homologues in the protein database. Some of the clones identified as apomictic-specific shared homology with known genes enabling their functional annotation. The relationships of these functions to the generation of the apomictic trait are discussed.     

Predicting gene function by conserved co-expression

We show that gene co-expression, which generally provides only a very weak signal for the prediction of functional interactions, can provide a reliable signal by exploiting evolutionary conservation. The encoded proteins of conserved co-expressed gene pairs are highly likely to be part of the same pathway not only after speciation (98%), but also after parallel gene duplication (97%). Conserved co-expression combined with homology data enables us to predict specific gene functions. The use of conservation between parallel duplicated gene pairs to predict function is especially promising given that gene duplication is common in eukaryotes, and that data from only a single organism can be used.     

Deletion of the SNARE vti1b in mice results in the loss of a single SNARE partner,syntaxin 8

SNARE proteins participate in recognition and fusion of membranes. A SNARE complex consisting of vti1b, syntaxin 8, syntaxin 7, and endobrevin/VAMP-8 which is required for fusion of late endosomes in vitro has been identified recently. Here, we generated mice deficient in vti1b to study the function of this protein in vivo. vti1b-deficient mice had reduced amounts of syntaxin 8 due to degradation of the syntaxin 8 protein, while the amounts of syntaxin 7 and endobrevin did not change. These data indicate that vti1b is specifically required for the stability of a single SNARE partner. vti1b-deficient mice were viable and fertile. Most vti1b-deficient mice were indistinguishable from wild-type mice and did not display defects in transport to the lysosome. However, 20% of the vti1b-deficient mice were smaller. Lysosomal degradation of an endocytosed protein was slightly delayed in hepatocytes derived from these mice. Multivesicular bodies and autophagic vacuoles accumulated in hepatocytes of some smaller vti1b-deficient mice. This suggests that other SNAREs can compensate for the reduction in syntaxin 8 and for the loss of vti1b in most mice even though vti1b shows only 30% amino acid identity with its closest relative.     

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.     

Complex evolution of gypsy in Drosophilid species

In an endeavor to contribute to the comprehension of the evolution of transposable elements (TEs) in the genome of host species, we investigated the phylogenetic relationships of sequences homologous to the retrotransposon gypsy of Drosophila melanogaster in 19 species of Drosophila, in Scaptodrosophila latifasciaeformis, and in Zaprionus indianus. This phylogenetic study was based on approximately 500 base pairs of the env gene. Our analyses showed considerable discrepancy between the phylogeny of gypsy elements and the relationship of their host species, and they allow us to infer a complex evolutionary pattern that could include ancestral polymorphism, vertical transmission, and several cases of horizontal transmission.     

Regulation of insulin-responsive aminopeptidase expression and targeting in the insulin-responsive vesicle compartment of glucose transporter isoform 4-deficient cardiomyocytes

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.     

Enhanced heart rate variability and baroreflex index after stress and cholinesterase inhibition in mice

Experiments tested the effect of stress coupled with cholinesterase inhibition on blood pressure, heart rate, baroreflex index, and variability in time and frequency domain in conscious mice. The objective was to determine whether cholinergic systems interact with stress to alter cardiovascular responses. Male C57BL/6J mice with arterial catheters were exposed to 3-day treatments: 1) intermittent shaker stress, 2) pyridostigmine (10 mg.kg(-1).day(-1)); or 3) combined pyridostigmine and stress. Pyridostigmine reduced blood cholinesterase (-33%) with no added effects of stress. Twenty-four-hour blood pressure recordings showed that there were no differences in blood pressure and heart rate with the treatments. Pulse interval standard deviation was greatly increased in the pyridostigmine/stress group compared with stress or pyridostigmine groups (11.0 +/- 1.4, 5.0 +/- 0.9, and 7.5 +/- 0.9 ms, respectively). Spectral analysis showed two distinct components for pulse interval variability (low and high frequency). Variability in the low-frequency range was greatly enhanced in the pyridostigmine/stress group, seen as a doubling of the power (9.5 +/- 1.7, 3.3 +/- 0.9, and 5.0 +/- 0.6 ms for pyridostigmine/stress, stress and pyridostigmine groups, respectively). Baroreflex sensitivity was also increased in the pyridostigmine/stress group (3.6 +/- 0.5 compared with 1.8 +/- 0.3 and 1.7 +/- 0.5 ms/mmHg in the stress and pyridostigmine groups, respectively). There was no difference in blood pressure variability or its spectral components. Results demonstrate that there are potent interactions between a mild stressor and cholinesterase inhibition seen as an accentuation of low-frequency variability in pulse interval time series, probably associated with baroreflex input and autonomic drive.     

Activity and mechanism of action of insect oostatic peptides in flesh fly

The relationship between structure and activity of insect oostatic decapeptide (Aed-TMOF) analogues in flesh fly was analyzed. The highest oostatic activity was exhibited by the pentapetide and tetrapeptide analogues, H-Tyr-Asp-Pro-Ala-Pro-OH and H-Tyr-Asp-Pro-Ala-OH, respectively. The tetrapeptide, either native or tritiated, was used to study its metabolism in the ovaries and hemolymph and to detect putative binding sites in the flesh fly ovaries and head. A high metabolism of the tetrapeptide with a half-life in the hemolymph and ovaries less than 1h was determined. The initial limiting step in the degradation is tyrosine(1) cleavage. Other degradation products were detected only transiently in low quantities. Using tritiated tetrapeptide, we found that only very low specific binding was detected in the homogenates of ovaries and in the rough membrane preparation in the presence and absence of protease inhibitors.