Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.     

In vitro Antifungal Activity of Essential oils and Major Components against Fungi Plant Pathogens

The indiscriminate use of synthetic fungicides has caused several problems to the environment, which place human and animal health at risk. Due to this fact, the search for natural alternatives to control phytopathogenic fungi growth has increased. This study aims to evaluate the antifungal activity of two essential oils (EOs) and three major components of EOs on the radial growth and spore germination of Fusarium verticillioides and Alternaria tenuissima. Minimum and half‐maximal inhibitory concentrations (CMI and CI₅₀) at 96 h for each treatment were calculated. Lemongrass EO and citral caused the highest inhibition for A. tenuissima (CMI of 1000 μl/l and CI₅₀ of 10 μl/l). For F. verticillioides, the most effective component was geraniol (CMI and CI₅₀ of 1000 and 250 μl/l, respectively). Spore germination rate was delayed by the EOs and major components tested. The use of EOs was effective to control these two fungal species in their different grow stages.     

Choice of land reference situation in life cycle impact assessment

Purpose

Land use life cycle impact assessment is calculated as a distance to target value—the target being a desirable situation defined as a reference situation in Milà i Canals et al.’s (Int J Life Cycle Assess 12(1):2–4, 2007) widely accepted framework. There are several reference situations. This work aims to demonstrate the effect of the choice of reference situation on land impact indicators.

Methods

Various reference situations are reported from the perspective of the object of assessment in land in life cycle assessment (LCA) studies and the modeling choices used in life cycle land impact indicators. They are analyzed and classified according to additional LCA modeling requirements: the type of LCA approach (attributional or consequential), cultural perspectives (egalitarian, hierarchist or individualist), and temporal preference. Sets of characterization factors (CF) by impact pathway, land cover, and region are calculated for different reference situations. These sets of CFs by reference situation are all compared with a baseline set. A case study on different crop types is used to calculate impact scores from different sets of CFs and compare them.

Results and discussion

Comparing the rankings of the CFs from two different sets present inversions from 5% to 35% worldwide. Impact scores of the case study present inversions of 10% worldwide. These inversions demonstrate that the choice of a reference situation may reverse the LCA conclusions for the land use impact category. Moreover, these reference situations must be consistent with the different modeling requirements of an LCA study (approach, cultural perspective, and time preference), as defined in the goal and scope.

Conclusions

A decision tree is proposed to guide the selection of a consistent and suitable choice of reference situation when setting other LCA modeling requirements.

     

Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K_m values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS. © 1996 Wiley-Liss, Inc.     

Porous devices derived from co-continuous polymer blends as a route for controlled drug release

In this study we examine the release profile of bovine serum albumin (BSA) from a porous polymer matrix derived from a co-continuous polymer blend. The porosity is generated through the selective extraction of one of the continuous phases. This is the first study to examine the approach of using morphologically tailored co-continuous polymer blends as a template for generating porous polymer materials for use in controlled release. A method for the preparation of polymeric capsules is introduced, and the effect of matrix pore size and surface area on the BSA release profile is investigated. Furthermore, the effect of surface charge on release is examined by surface modification of the porous substrate using layer-by-layer deposition techniques. Synthetic, nonerodible polymer, high-density polyethylene (HDPE), was used as a model substrate prepared by melt blending with two different styrene-ethylene-butylene copolymers. Blends with HDPE allow for the preparation of porous substrates with small pore sizes (300 and 600 nm). A blend of polylactide (PLA) and polystyrene was also used to prepare porous PLA with a larger pore size (1.5 microm). The extents of interconnectivity, surface area, and pore dimension of the prepared porous substrates were examined via gravimetric solvent extraction, BET nitrogen adsorption, mercury porosimetry, and image analysis of scanning electron microscopy micrographs. With a loading protocol into the porous HDPE and PLA involving the alternate application of pressure and vacuum, it is shown that virtually the entire porous network was accessible to BSA loading, and loading efficiencies of between 80% and 96% were obtained depending on the pore size of the carrier and the applied pressure. The release profile of BSA from the microporous structure was monitored by UV spectrophotometry. The influence of pore size, surface area, surface charge, and number of deposited layers is demonstrated. It is shown that an effective closed-cell structure in porous PLA can be prepared, effectively eliminating all short-term BSA release.     

Mitotic activity of the duodenal crypt enterocytes in mice transplanted with EA21a mammary carcinoma.

The presence of a tumor generally changes the mitotic activity of the normal cell population in mice. In the present work, the mitotic activity of the duodenal crypt enterocytes in EA21a mammary carcinoma-bearing mice was determined. The results show that there is a patent circadian variation in normal mice and, in the presence of the EA21a mammary tumor, cell proliferation is stimulated. Stimulation was evident in enterocytes from the intermediate as well as the superficial regions of the crypt. Some humoral factors produced by the transplanted tumor could interfere with the regulatory mechanism of the mitotic activity of duodenal crypt enterocytes.     

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn’t recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3–6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3–6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3–6 hairpin than the wild-type hairpin. The SΔ1Δ3–6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30–37 days, while mice infected with SΔ1Δ3–6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV “flip and flop” telomeres are arranged.     

Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.     

Beyond AICA riboside: in search of new specific AMP-activated protein kinase activators

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects.