An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 (^1-78REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated ^1-78REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy.     

Structural characteristics of the plasmid-encoded toxin from enteroaggregative Escherichia coli

Intoxication by the plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli requires toxin translocation from the endoplasmic reticulum (ER) to the cytosol. This event involves the quality control system of ER-associated degradation (ERAD), but the molecular details of the process are poorly characterized. For many structurally distinct AB-type toxins, ERAD-mediated translocation is triggered by the spontaneous unfolding of a thermally unstable A chain. Here we show that Pet, a non-AB toxin, engages ERAD by a different mechanism that does not involve thermal unfolding. Circular dichroism and fluorescence spectroscopy measurements demonstrated that Pet maintains most of its secondary and tertiary structural features at 37 degrees C, with significant thermal unfolding only occurring at temperatures >or=50 degrees C. Fluorescence quenching experiments detected the partial solvent exposure of Pet aromatic amino acid residues at 37 degrees C, and a cell-based assay suggested that these changes could activate an ERAD-related event known as the unfolded protein response. We also found that HEp-2 cells were resistant to Pet intoxication when incubated with glycerol, a protein stabilizer. Altogether, our data are consistent with a model in which ERAD activity is triggered by a subtle structural destabilization of Pet and the exposure of Pet hydrophobic residues at physiological temperature. This was further supported by computer modeling analysis, which identified a surface-exposed hydrophobic loop among other accessible nonpolar residues in Pet. From our data it appears that Pet can promote its ERAD-mediated translocation into the cytosol by a distinct mechanism involving partial exposure of hydrophobic residues rather than the substantial unfolding observed for certain AB toxins.     

Chemical composition of the sponge Chondrosia reniformis from [2pt] the Canary Islands

Lake Zempoala was studied throughout 16 months in 1996–1997. It is a shallow monomictic lake situated at 2800 masl at the Neovolcanic Belt, well within the Mexican tropical zone. Most of the phytoplankton species in this lake may be characterized as temperate, according to their geographical distribution. A break down in phytoplankton biomass was observed before the lake's circulation, and open to question if a clear-water phase could be present in a tropical lake.     

p32 (gC1qBP) is a general protein kinase C (PKC)-binding protein; interaction and cellular localization of P32-PKC complexes in ray hepatocytes.

The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homogenates. The protein was identified by NH(2)-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC activators was different for each one. Whereas PKCdelta only binds to p32 in the presence of PKC activators, PKCzeta and PKCalpha increase their binding when they are in the activated form. Other PKC isoforms such as beta, epsilon, and theta bind equally well to p32 regardless of the presence of PKC activators, and PKCmu binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKCdelta) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal microscopy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKCtheta and p32 are constitutively associated in vivo, whereas PKCdelta activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.     

The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication

A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M(r), of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed in Escherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.     

Cell-mediated immune responses (CMIR) in human rhinosporidiosis

Cell mediated immune responses (CMIR) to Rhinosporidium seeberi in human patients with rhinosporidiosis have been studied. With immuno-histochemistry, the cell infiltration patterns in rhinosporidial tissues from 7 patients were similar. The mixed cell infiltrate consisted of many plasma cells, fewer CD68+ macrophages,a population of CD3+ T lymphocytes, and CD56/57+ NK lymphocytes which were positive for CD3 as well. CD4+ T helper cells were scarce. CD8+suppressor/cytotoxic-cytolytic cells were numerous. Most of the CD8+ cells were TIA-l+ and therefore of the cytotoxic subtype. CD8+ T cells were not sub-typed according to their cytokine profile; 1L2, IFN-γ (Tcl); IL4, ILS (Tc2).In lympho-proliferative response (LPR) assays in vitro, lymphocytes from rhinosporidial patients showed stimulatory responses to Con A but lymphocytes from some patients showed significantly diminished responses to rhinosporidial extracts as compared with unstimulated cells or cells stimulated by Con A, indicating suppressor immune responses in rhinosporidiosis. The overall stimulatory responses with Con A suggested that the rhinosporidial lymphocytes were not non-specifically anergic although comparisons of depressed LPR of rhinosporidial lymphocytes from individual patients, to rhinosporidial antigen with those to Con A, did not reveal a clear indication as to whether the depression was antigen specific or non-specific. The intensity of depression of the LPR in rhinosporidial patients bore no relation to the site, duration, or the number of lesions or whether the disease was localized or disseminated. Rhinosporidial extracts showed stimulatory activity on normal control lymphocytes, perhaps indicating mitogenic activity. These results indicate that CMIR develops in human rhinosporidiosis, while suppressed responses are also induced. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cranial ontogeny of<Emphasis Type="Italic">Lutreolina crassicaudata</Emphasis> (Didelphidae): a comparison with<Emphasis Type="Italic">Didelphis albiventris</Emphasis>

Metatherians experience the greatest developmental changes during extrauterine life. Following previous studies onDidelphis albiventris Lund, 1840, we examined the postweaning cranial allometry of size of the Neotropical marsupialLutreolina crassicaudata (Desmerest, 1804). Our aim was to compare growth patterns of both species to identify traits particular to each species and traits common to both species. This may contribute toward identifying a common developmental plan for didelphids. We measured 15 cranial variables in 32–43 specimens from just-weaned young to old adult. Total length of the skull was the estimator of overall size in least squares and reduced major axis regressions. The skull ofLutreolina crassicaudata grows at a rate slower than the overall change in size in its neurocranial components, palate, and postcanine rows, and it grows relatively faster in the rest of the splanchnocranium. This pattern closely resembles that ofDidelphis albiventris, from which it differs mainly in the allometry of the muzzle. In both species, allometry explains most postweaning changes of the trophic apparatus on functional grounds, in relation to interspecific differences in diet. We hypothesize that most local allometric departures from a generalized didelphid plan would relate to main dietary trends.