Synthesis and cytotoxicity of hydrophobic esters of podophyllotoxins

Diverse norbornenecarboxylate esters of podophyllotoxin and its epimers and diastereoisomers have been prepared through Diels-Alder cycloaddition by treating the dienophilic acrylates of cyclolignans with cyclopentadiene. Their cytotoxicities against several cancer cell lines have been evaluated and the results compared with those found for other lignan esters. Podophyllotoxin adducts showed a one-fold increase in activity when compared to the natural product. The preparation of more hydrophobic esters, which showed less cytotoxicity, demonstrated that this activity is not primarily due to the lipophilic factor, but mainly to the spatial arrangement of the bulky moiety, which could contribute to increase the binding to the target site.     

Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments

The organization of secretory traffic remains unclear, mainly because of the complex structure and dynamics of the secretory pathway. We have thus studied a simplified system, a single synchronized traffic wave crossing an individual Golgi stack, using electron tomography. Endoplasmic-reticulum-to-Golgi carriers join the stack by fusing with cis cisternae and induce the formation of intercisternal tubules, through which they redistribute their contents throughout the stack. These tubules seem to be pervious to Golgi enzymes, whereas Golgi vesicles are depleted of both enzymes and cargo. Cargo then traverses the stack without leaving the cisternal lumen. When cargo exits the stack, intercisternal connections disappear. These findings provide a new view of secretory traffic that includes dynamic intercompartment continuities as key players.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.     

Combined treatment with vessel dilator and kaliuretic hormone in persons with congestive heart failure

Vessel dilator and kaliuretic hormone, two cardiovascular peptide hormones, enhance urine flow 2- to 13-fold and 4-fold, respectively, in persons with class III New York Heart Association congestive heart failure (CHF). The natriuresis and diuresis secondary to vessel dilator and kaliuretic hormone are not blunted as are atrial natriuretic peptide and brain natriuretic peptide effects in persons with CHF compared with healthy individuals. The present investigation determined if the two peptide hormones that do not have blunted effects in persons with CHF may have added beneficial effects when given simultaneously to individuals with class III CHF. Together with each at 100 ng/kg of body weight per minute, vessel dilator and kaliuretic hormone increased urine flow rate 3.5-fold (P < 0.05) compared with their 60-min baseline and control CHF subjects' urine flow rates. Combined, they enhanced the excretion rate of sodium a maximum of 3.6-fold (P < 0.05) with 2.5- and 2-fold enhancement 2 and 3 hrs after infusion. These data indicate that vessel dilator and kaliuretic hormone have diuretic and natriuretic effects when used in combination, but these effects are not additive over their individual effects in persons with CHF.     

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.     

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.     

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.