SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.     

First Homo erectus from Turkey and implications for migrations into temperate Eurasia

Remains of fossil hominins from temperate regions of the Old World are rare across both time and space, but such specimens are necessary for understanding basic issues in human evolution including linkages between their adaptations and early migration patterns. We report here the remarkable circumstances surrounding the discovery of the first fossil hominin calvaria from Turkey. The specimen was found in the Denizli province of western Turkey and recovered from within a solid block of travertine stone as it was being sawed into tile-sized slabs for the commercial natural stone building market. The new specimen fills an important geographical and temporal gap and displays several anatomical features that are shared with other Middle Pleistocene hominins from both Africa and Asia attributed to Homo erectus. It also preserves an unusual pathology on the endocranial surface of the frontal bone that is consistent with a diagnosis of Leptomeningitis tuberculosa (TB), and this evidence represents the most ancient example of this disease known for a fossil human. TB is exacerbated in dark-skinned peoples living in northern latitudes by a vitamin D deficiency because of reduced levels of ultraviolet radiation (UVR). Evidence for TB in the new specimen supports the thesis that reduced UVR was one of the many climatic variables presenting an adaptive challenge to ancient hominins during their migration into the temperate regions of Europe and Asia.     

Effect of stress-induced lipid peroxidation on functions of rat peritoneal macrophages

The aim of the present study was to investigate the effects of stress-induced lipid peroxidation on macrophages' functions. Animals were subjected to 4 h immobilization at 4 degrees C in restraining devices. The peritoneal macrophages obtained from rats exposed to cold and restraint stress exhibited an increase in lipid peroxidation and a decline of chemotaxis and phagocytosis compared with control rats. After supplementation with vitamin E, the increment in thiobarbituric acid reactive substances (TBARS) content as the oxidative stress marker and the decline of chemotaxis and phagocytosis in peritoneal macrophages observed during cold-restraint stress was significantly removed. No significant change in catalase activity of peritoneal macrophages was observed in groups exposed to cold-restraint stress and treated with vitamin E. These findings indicate that phagocytic and chemotactic capacities of peritoneal macrophages are decreased by cold-restraint stress and this effect of stress may be related to lipid peroxidation.     

Chemical composition effects onto antimicrobial and antioxidant activities of propolis collected from different regions of Turkey

Chrysin, apigenin, flavonoids, flavanones, naringenin, ethyl oleate, 3-4-dimethoxy-cinnamic acid and 9-octadecenoic acid were the predominant components of propolis samples collected from different regions of Turkey. The extracts of P3 from Denizli-Ba?karci, P5 from Denizli and P7 from Tekirda? had effective antibacterial activities on Gram-negatives. Chrysin, which has antibacterial activity, was found to be high concentration. The extracts of P3, P2B from Aydin and P6 from Konya had much more effective antibacterial activities on Gram-positives. The total antioxidant activity increased with the increasing amount of extracts added to linoleic acid emulsion. All doses of propolis ethanol extract displayed antioxidant activity.     

Risk Attitudes of Organic and Conventional Hazelnut Producers in Turkey

Understanding farmers’ attitudes and responses to agricultural risks is important for designing financial support services and effective extension activities. Risk attitudes of farmers are also one of the critical barriers for conversion to organic agriculture. Few studies have been conducted to explore and compare risk perceptions between organic and conventional farmers. The purposes of this research were to elicit and compare the risk attitudes of organic and conventional Turkish hazelnut producers and to determine the barriers additional to their risk attitudes toward conversion to organic agriculture in the Terme district of Samsun, Turkey. The Equally Likely Certainty Equivalent (ELCE) model was used to elicit responses from producers. The data used in this study were collected by using a structured questionnaire administered to both 64 randomly selected conventional hazelnut producers and all organic hazelnut producers (39) in the research area. Research results revealed that organic hazelnut producers were less risk averse than conventional producers. It was also found that the variables of education level of operators, farm income, total assets, time allocated outside the farm, farm land area, size of hazelnut orchard, number of hazelnut plots, and total information score were other barriers for conversion to organic hazelnut production, in addition to the producers’ risk attitudes. Providing better extension services and farmers’ training activities relating to organic agriculture and land consolidation, focusing on raising the educational level of producers, facilitating producers with greater access to credit to increase farm land and to enhance farm assets are factors that may accelerate the process of conversion to organic hazelnut production.     

Analyzing the functions of large glycoconjugates through the dissipative properties of their absorbed layers using the gel-forming mucin MUC5B as an example

Glyconjugates such as mucins, proteoglycans, and polysaccharides form the structural basis of protective cell-surface layers. In particular gel-forming mucins define a zone between the epithelial cell layer and the environment. Such molecules are of extreme molecular weight 5-100 x 10(6) and size (Rg 20-300 nm). On this account their biochemistry is inseparable from their physical biochemistry. Combining laser light scattering and quartz crystal mass balance with dissipation methods (QCM-D) we have investigated the properties of the MUC5B mucin and its cognate fragments when bound to a hydrophobic surface. MUC5B forms the basis of gels responsible for the protection of the oral cavity, lung, and cervical canal surfaces. Here we show, by analyzing dissipative interactions of hydrophobic, gold, and polystyrene surfaces, with the intact MUC5B molecule, its reduced subunits, and glycosylated tryptic fragments (obtained after reduction), the formation of 40- to 100-nm-thick highly structured, hydrated interfaces. These interfaces are dominated in their geometry and dissipative properties by the negatively charged carbohydrate-rich domains of the molecule, the naked protein domains being responsible for attachment. These carbohydrate-rich surfaces have well-defined absorptive properties and permit the entry and entrapment of albumin-coated micro-beads into the absorbed layer at and below a size of 60 nm. However beads larger than 100 nm are completely excluded from the surfaces. These absorptive phenomena correlate with large changes in film dissipation and thus may not only be important in biological functions, e.g. binding viruses, but could also be informative to the surfaces (often ciliated) onto which such mucus films are attached.     

Metabolic preconditioning of cells with AICAR-riboside: Improved cryopreservation and cell-type specific impacts on energetics and proliferation

In species whose evolutionary history has provided natural tolerance to dehydration and freezing, metabolic depression is often a pre-requisite for survival. We tested the hypothesis that preconditioning of mammalian cells with 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside (AICAR) to achieve metabolic depression will promote greater survivorship during cryopreservation. AICAR is used extensively to stimulate AMP-activated protein kinase (AMPK), which can result in downregulation of biosynthetic processes. We showed that the metabolic interconversion of AICAR was cell-type dependent. Accumulation of 5-aminoimidazole-4-carboxamide-1b-d-ribofuranosyl-5′-monophosphate (ZMP), as well as other metabolites that possess multiple phosphates (i.e., ZDP, ZTP), varied approximately 3.5-fold across the cell lines tested. AICAR treatment also significantly influenced the concentrations of cellular adenylates (ATP, ADP, and AMP). Depression of cell metabolism and proliferation with AICAR treatment differed among cell lines. Proliferation for a given cell line was negatively correlated with the fold-increase achieved in the ‘effective adenylate ratio’ ([AMP] + [ZMP])/[ATP]) after AICAR treatment. Metabolic preconditioning with AICAR promoted a significant increase in viability post-freezing in J774.A1 macrophages, HepG2/C3A cells and primary hepatocytes but not in NIH/3T3 fibroblasts or OMK cells. The effect of AICAR on viability after freezing was positively correlated (r² = 0.94) with the fold-increase in the ‘effective adenylate ratio’. Thus for each cell line, the greater the depression of metabolism and proliferation due to preconditioning with AICAR, the greater was the survivorship post-freezing.