Evolutionary history of chromosome 10 in primates

We have tracked the evolutionary history of chromosomes homologous to HSA10 (PHYL-10) in primates using appropriate panels of PCP, YAC, and BAC probes. This approach allowed us to delineate more precisely the PHYL-10 constitution in the ancestor of catarrhine, platyrrhine, and prosimians. The results suggest that (i) in the ancestor of prosimians PHYL-10 was organized in two separate PHYL-10p and PHYL-10q chromosomes; (ii) in the progenitor of New World monkeys PHYL-10p was a separate chromosome, while PHYL-10q was associated with a chromosome homologous to HSA16; (iii) in the ancestor of Old World monkeys PHYL-10 was a unique chromosome with a marker order corresponding to the orang form. We have also analyzed the cat, chosen as an outgroup for its very conserved karyotype. In agreement with published data our experiments show that the PHYL-10 in cat is structured in two blocks, PHYL-10p and PHYL-10q, both as part of larger chromosomes. The overall data indicate that, contrary to common opinion, PHYL-10p and PHYL-10q were distinct chromosomes in the primate ancestor. Analysis of the Saimiri sciureus (SSC) PHYL-10q marker order showed that it was isosequential with the Callithrix jacchus PHYL-10q, as well as with the PHYL-10q platyrrhine ancestral form. The SSC centromere, nevertheless, was located in a different chromosomal region, therefore suggesting that a centromeric repositioning event occurred in this species.     

The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with,but not necessary for,GRP 78-mediated signal transduction

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.     

Hantavirus pulmonary syndrome in Uberaba,Minas Gerais,Brazil

This report describes the epidemiological and clinical-evolutive characteristics of eight patients with hantavirus pulmonary syndrome (HPS) in Uberaba, Minas Gerais, Brazil. A positive history of contact with rodents was present in 100% of the cases. The time between the onset of symptoms and hospital care was, on average, 3.6 days. All patients showed clinical and laboratory findings suggestive of HPS. Elevated urea and creatinine levels were observed in 6 (75%) cases, PO2 was < 60 mmHg in 100% of the cases, and a chest X-ray demonstrated a bilateral interstitial-alveolar infiltrate. The diagnosis was confirmed by the detection of IgM antibodies against Sin Nombre virus by ELISA. Three patients died as a direct consequence of HPS.     

Antisense down regulation of NtBI-1 in tobacco BY-2 cells induces accelerated cell death upon carbon starvation

Bax inhibitor-1 (BI-1) protein is proposed to be a conserved programmed cell death suppressor. In this report, we investigate the anti-apoptotic function of plant BI-1 by antisense (AS) down regulation of NtBI-1 in Nicotiana tabacum cv. BY-2 cells. We observed that AS cell lines were more susceptible to autophagy, internucleosomal DNA fragmentation and death than control cells when subjected to sucrose starvation and hypo-osmotic shock, in agreement with a role of BI-1 as a death inhibitor.     

Late endosome motility depends on lipids via the small GTPase Rab7

We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.     

Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones

Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.     

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.     

Structure and dynamics of a helical hairpin and loop region in annexin 12: a site-directed spin labeling study

A 30-residue nitroxide scan encompassing a helical hairpin and an extended loop in soluble annexin 12 (helices D and E in repeat 2; residues 134-163) has been analyzed in terms of nitroxide side chain mobility and accessibility to collision with paramagnetic reagents (Pi). Values of Pi for both O(2) and a Ni(II) metal complex (NiEDDA) are remarkably well correlated with the fractional solvent accessibility of the native side chains at the corresponding positions computed from the known crystal structure. This result demonstrates the utility of Pi as an experimental measure of side chain accessibility in solution, as well as the lack of structural perturbation due to the presence of the nitroxide side chain. The pattern of side chain mobility is also in excellent agreement with predictions from the crystal structure. The results presented here extend the correlations between mobility and structure described in earlier work on other helical proteins, and suggest their generality. The periodic dependence of Pi and mobility along the sequence of annexin 12 reveals the helical segments and their orientation in the fold, as expected for a nonperturbing nitroxide side chain. However, these data do not distinguish the helix-loop-helix motif from a continuous helix, because immobilized side chains in the short loop sequence maintain the periodicity. As shown here, the ratio of Pi values for O(2) and NiEDDA clearly delineates the loop region, due to size exclusion effects between the two reagents. A new feature evident in a nitroxide scan through multiple secondary elements is a modulation of the basic Pi and mobility patterns along the sequence, apparently due to differences in helix packing and backbone motion. Thus, in the short helix D, residues are consistently more mobile and accessible throughout the sequence compared to the residues in the longer, less-solvated and more ordered helix E.