Colloidal aggregation affects the efficacy of anticancer drugs in cell culture

Many small molecules, including bioactive molecules and approved drugs, spontaneously form colloidal aggregates in aqueous solution at micromolar concentrations. Though it is widely accepted that aggregation leads to artifacts in screens for ligands of soluble proteins, the effects of colloid formation in cell-based assays have not been studied. Here, seven anticancer drugs and one diagnostic reagent were found to form colloids in both biochemical buffer and in cell culture media. In cell-based assays, the antiproliferative activities of three of the drugs were substantially reduced when in colloidal form as compared to monomeric form; a new formulation method ensured the presence of drug colloids versus drug monomers in solution. We also found that Evans Blue, a dye classically used to measure vascular permeability and to demonstrate the "enhanced permeability and retention (EPR) effect" in solid tumors, forms colloids that adsorb albumin, as opposed to older literature that suggested the reverse.     

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

Movements of wintering surf scoters: predator responses to different prey landscapes

The distribution of predators is widely recognized to be intimately linked to the distribution of their prey. Foraging theory suggests that predators will modify their behaviors, including movements, to optimize net energy intake when faced with variation in prey attributes or abundance. While many studies have documented changes in movement patterns of animals in response to temporal changes in food, very few have contrasted movements of a single predator species naturally occurring in dramatically different prey landscapes. We documented variation in the winter movements, foraging range size, site fidelity, and distribution patterns of a molluscivorous sea duck, the surf scoter (Melanitta perspicillata), in two areas of coastal British Columbia with very different shellfish prey features. Baynes Sound has extensive tidal flats with abundant clams, which are high-quality and temporally stable prey for scoters. Malaspina Inlet is a rocky fjord-like inlet where scoters consume mussels that are superabundant and easily accessible in some patches but are heavily depleted over the course of winter. We used radio telemetry to track surf scoter movements in both areas and found that in the clam habitats of Baynes Sound, surf scoters exhibited limited movement, small winter ranges, strong foraging site fidelity, and very consistent distribution patterns. By contrast, in mussel habitats in the Malaspina Inlet, surf scoters displayed more movement, larger ranges, little fidelity to specific foraging sites, and more variable distribution patterns. We conclude that features associated with the different prey types, particularly the higher depletion rates of mussels, strongly influenced seasonal space use patterns. These findings are consistent with foraging theory and confirm that predator behavior, specifically movements, is environmentally mediated.     

First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile

Background  

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).     

Weight regain and health-related quality of life in postmenopausal women

Weight loss improves health-related quality of life (HRQoL). However, regain after loss is common; little is known about the impact of weight regain on HRQoL in postmenopausal women. Woman on the Move through Activity and Nutrition (WOMAN) is a randomized lifestyle intervention trial of diet, physical activity, and weight loss in 508 postmenopausal women aged 52-62 years. This analysis focused on the women who lost > or =5 lb during the initial phase of the study, baseline to 6 months (n = 248). This cohort was divided into three groups based on subsequent weight change between 6 and 18 months: weight loss (WL; > or =5 lb loss), weight stable (WS; <+/-5 lb change), and weight regain (WR; > or =5 lb gain). HRQoL was measured at baseline, 6, and 18 months using the Short Form-36. Of the 248 women studied, 51 (21%) continued to lose weight after initial weight loss, while 127 (51%) maintained a stable weight, and 70 (28%) regained weight. Between baseline and 6 months, women in WR group had decreased mental health and social-functioning scores, while the WL and WS groups improved in these subscales. Between baseline and 18 months, energy improved most significantly in those with continued weight loss (P = 0.0003). Weight loss was correlated with a small to moderate improvement in perceived general health and energy, which was reversed by weight gain. Further study is needed to investigate the impact of a decline in mental health and social functioning on future weight regain.     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

Spectral monitoring of surfactant clearance during alveolar epithelial type II cell differentiation

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment.     

Molecular mapping of the C locus for presence of pungency in Capsicum.

Pungency owing to the presence of capsaicinoids is a unique character of pepper (Capsicum spp.). Capsaicinoids are produced in the placenta and it has long been known that a single dominant gene, C, is required for pungent genotypes to produce capsaicinoids. We mapped C to pepper chromosome 2 in a cross between a pungent Capsicum frutescens wild accession and a non-pungent Capsicum annuum bell pepper. This position confirmed results from earlier studies. The RFLP marker TG 205 cosegregated with C and two additional RFLP markers were also located within 1 cM. The recessive allele at the C locus is used in breeding programs around the world focused on very diverse germplasm, hence any of these tightly linked markers may be of value as potential sources of useful markers for marker-assisted selection. To demonstrate this point, we developed a PCR-based CAPS (cleaved amplified polymorphic sequence) marker linked to C using the sequence of the Capsicum fibrillin gene located 0.4 cM from C. The use of molecular markers for high-throughput screening for the c allele in pepper breeding programs is discussed.     

Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo

Summary Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)₂ fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)₂ fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)₂ compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)₂antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)₂antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)₂AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)₂ fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)₂AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)₂AELG (0.0052AELG MTX linked to the F(ab)₂ fragment of AELG - MTX-F(ab)₂antiBSA MTX linked to the F(ab)₂ fragment of antiBSA - MTX-F(ab)₂NRG MTX linked to the F(ab)₂ fragment of NRG - MTX-NRG MTX linked to NRG - NHS N-hydroxysuccinimide - NRG normal rabbit IgG - PBS 0.01 M sodium phosphate (pH 7.1) containing 0.45 M sodium chloride - TAA tumor-associated antigen - t_1/2 half-life