Molecular distinction between pathogenic and infectious properties of the prion protein

Tg(PG14) mice express a prion protein (PrP) with a nine-octapeptide insertion associated with a human familial prion disease. These animals spontaneously develop a fatal neurodegenerative disorder characterized by ataxia, neuronal apoptosis, and accumulation in the brain of an aggregated and weakly protease-resistant form of mutant PrP (designated PG14(spon)). Brain homogenates from Tg(PG14) mice fail to transmit disease after intracerebral inoculation into recipient mice, indicating that PG14(spon), although pathogenic, is distinct from PrP(Sc), the infectious form of PrP. In contrast, inoculation of Tg(PG14) mice with exogenous prions of the RML strain induces accumulation of PG14(RML), a PrP(Sc) form of the mutant protein that is infectious and highly protease resistant. Like PrP(Sc), both PG14(spon) and PG14(RML) display conformationally masked epitopes in the central and octapeptide repeat regions. However, these two forms differ profoundly in their oligomeric states, with PG14(RML) aggregates being much larger and more resistant to dissociation. Our analysis provides new molecular insight into an emerging puzzle in prion biology, the discrepancy between the infectious and neurotoxic properties of PrP.     

Angiogenesis in collagen I requires alpha2beta1 ligation of a GFP*GER sequence and possibly p38 MAPK activation and focal adhesion disassembly

Angiogenesis depends on proper collagen biosynthesis and cross-linking, and type I collagen is an ideal angiogenic scaffold, although its mechanism is unknown. We examined angiogenesis using an assay wherein confluent monolayers of human umbilical vein endothelial cells were overlain with collagen in a serum-free defined medium. Small spaces formed in the cell layer by 2 h, and cells formed net-like arrays by 6-8 h and capillary-like lumens by 24 h. Blocking of alpha2beta1, but not alpha1 or alpha(v)beta3 integrin function halted morphogenesis. We found that a triple-helical, homotrimeric peptide mimetic of a putative alpha2beta1 binding site: alpha1(I)496-507 GARGERGFP*GER (where single-letter amino acid nomenclature is used, P* = hydroxyproline) inhibited tube formation, whereas a peptide carrying another putative site: alpha1(I)127-138 GLP*GERGRP*GAP* or control peptides did not. A chemical inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), SB202190, blocked tube formation, and p38 MAPK activity was increased in collagen-treated cultures, whereas targeting MAPK kinase (MEK), focal adhesion kinase (FAK), or phosphatidylinositol 3-kinase (PI3K) had little effect. Collagen-treated cells had fewer focal adhesions and 3- to 5-fold less activated FAK. Thus capillary morphogenesis requires endothelial alpha2beta1 integrin engagement of a single type I collagen integrin-binding site, possibly signaling via p38 MAPK and focal adhesion disassembly/FAK inactivation.     

Identification of an autoinhibitory domain of p21-activated protein kinase 5

The p21-activated protein kinases (Paks) are serine/threonine protein kinases activated by binding to Rho family small GTPases, Rac and Cdc42. Recently, Pak family members have been subdivided into two groups, I and II. Group II Paks, including Pak4, Pak5, and Pak6, does not contain the highly conserved autoinhibitory domain that is found in the group I Paks members, i.e. Pak1, Pak2, and Pak3. In the present study, we have purified the glutathione S-transferase fusion form of Pak5 and shown for the first time that Pak5 autophosphorylation can be activated by GTP bound form of Cdc42. Mutation of histidine residues 19 and 22 to leucine on the p21-binding domain of Pak5 completely abolished the binding of Cdc42 and the Cdc42-mediated autophosphorylation. On the other hand, mutation of tyrosine 40 to cysteine of Cdc42 did not knockout the binding of Pak5. Analysis of C-terminal deletion mutants has identified an autoinhibitory fragment of Pak5 that is absent from other group II Pak family members. Taken together, these results suggest that Pak5, like Pak1, contains an autoinhibitory domain and its activity is regulated by Cdc42.     

Cloning,expression, characterization,and role in autocrine cell growth of cell surface retention sequence binding protein-1

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Pharmaco-toxicological study of Kageneckia oblonga, Rosaceae

The probable antipyretic, antiinflammatory, analgesic and antioxidant properties of Kageneckia oblonga, Rosaceae, were investigated and the major compounds of its active extracts were isolated. The study comprised the acute toxicity of the extracts of global methanol, hexane, dichloromethane and methanol. The cytotoxicity of global methanol extract was studied in three tumoral cell lines. All the extracts exhibited the pharmacological activities under study. Methanol and dichloromethane were the most toxic extracts. From the global methanol extract, isolations were performed of prunasin, 23,24- dihydro-cucurbitacin F, and a new cucurbitacin, 3beta-(beta-D-glucosyloxy)-16alpha,23alpha-epoxycucurbita-5,24-diene-11-one. The cytotoxicity of both cucurbitacins on human neutrophils at the assayed concentrations was not statistically significant. In-vitro assays showed that both cucurbitacins can be partly responsible for the analgesic, antipyretic, and anti-inflammatory activities. Evaluation was done of the cytotoxicity of global methanol extract, 23, 24-dihydrocucurbitacin F, aqueous extracts and prunasin against P-388 murine leukaemia, A-549 human lung carcinoma and HT-29 colon carcinoma. Since global methanol extract presented a strong cytotoxicity against P-388 murine leukaemia, A-549 human lung carcinoma, and HT-29 cell lines, it is highly probable that this extract contain one or more cytotoxic compounds that could be investigated for their potential use as an agent against cancer.