Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Studies on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor and thermolysin.

The effects of certain physicochemical parameters on the formation and stability of a complex between Streptomyces proteinaceous metalloprotease inhibitor (SMPI) and thermolysin were investigated. SMPI had its lowest Ki value at a pH of around 6.5 (similar to the pH dependence of the kcat/K(m) of thermolysin catalysis), reflecting the splitting mechanism of the SMPI inhibition of thermolysin. This Ki increased with an increase in pressure, and in (Ki-1) was almost linear with respect to pressure. The volume of the reaction (delta Vcomp), which is the volume change accompanying enzyme-inhibitor complex formation, was calculated as +8.1 +/- 0.3 mL.mol-1, which has a sign opposite to delta Vcomp for neutral peptide inhibitors and acyl-peptide substrates. The temperature dependence of Ki-1 gave the reaction enthalpy (delta Hcomp) and reaction entropy (delta Scomp) of the complex formation as 34.6 +/- 1.4 kJ.mol-1 and 298 +/- 5 J.mol-1.K-1, respectively. These positive reaction volumes and reaction entropies were related to the electrostatic interactions and ionic strength dependence of Ki which corresponded to the key ionic interaction during complex formation. Complex formation with SMPI stabilized thermolysin against pressure perturbation as observed by the changes in the Trp fluorescence of thermolysin with increasing pressure. Thermal stability, however, was affected very little by complex formation with SMPI. Phosphoramidon, Cbz-Phe-Gly-NH2 and Cbz-Phe also positively affected the pressure-tolerance of thermolysin, in the following order: Cbz-Gly-Phe-NH2 < Cbz-Phe < phosphoramidon. The third compound exhibited stabilizing effects comparable with those of SMPI, which suggests that the interaction between SMPI and thermolysin was localized to the reactive site.     

The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.     

Do you perform the multiple sleep latency test according to the guidelines? A case with multiple sleep onset REM periods

Sleep and Biological Rhythms - The patient was a 21-year-old male who complained of daytime sleepiness. His multiple sleep latency test (MSLT) showed multiple sleep onset REM periods (SOREMPs). The...     

BAG-6 is essential for selective elimination of defective proteasomal substrates

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.     

PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.     

The Spemann organizer meets the anterior‐most neuroectoderm at the equator of early gastrulae in amphibian species

The dorsal blastopore lip (known as the Spemann organizer) is important for making the body plan in amphibian gastrulation. The organizer is believed to involute inward and migrate animally to make physical contact with the prospective head neuroectoderm at the blastocoel roof of mid‐ to late‐gastrula. However, we found that this physical contact was already established at the equatorial region of very early gastrula in a wide variety of amphibian species. Here we propose a unified model of amphibian gastrulation movement. In the model, the organizer is present at the blastocoel roof of blastulae, moves vegetally to locate at the region that lies from the blastocoel floor to the dorsal lip at the onset of gastrulation. The organizer located at the blastocoel floor contributes to the anterior axial mesoderm including the prechordal plate, and the organizer at the dorsal lip ends up as the posterior axial mesoderm. During the early step of gastrulation, the anterior organizer moves to establish the physical contact with the prospective neuroectoderm through the “subduction and zippering” movements. Subduction makes a trench between the anterior organizer and the prospective neuroectoderm, and the tissues face each other via the trench. Zippering movement, with forming Brachet's cleft, gradually closes the gap to establish the contact between them. The contact is completed at the equator of early gastrulae and it continues throughout the gastrulation. After the contact is established, the dorsal axis is formed posteriorly, but not anteriorly. The model also implies the possibility of constructing a common model of gastrulation among chordate species.