Creating biological membranes on the micron scale: forming patterned lipid bilayers using a polymer lift-off technique

We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 microm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 micro m-76 microm on edge, provide a synthetic biological substrate for biochemical analysis that is approximately 100x smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC(18)(3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 +/- 1.25 microm(2) s(-1), equal to or slightly faster than typically found in DiI stained cells. This diffusion rate is approximately 3x faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation.     

A new species of Mesocoelium (Digenea: Mesocoeliidae) found in Rhinella marina (Amphibia: Bufonidae) from Brazilian Amazonia

Mesocoelium lanfrediaesp. nov. (Digenea: Mesocoeliidae) inhabits the small intestine of Rhinella marina (Amphibia: Bufonidae) and is described here, with illustrations provided by light, scanning electron microscopy and molecular approachs. M. lanfrediae sp. nov. presents the typical characteristics of the genus, but is morphometrically and morphologically different from the species described previously. The main diagnostic characteristics of M. lanfrediae sp. nov. are (i) seven pairs of regularly-distributed spherical papillae on the oral sucker, (ii) ventral sucker outlined by four pairs of papillae distributed in a uniform pattern and interspersed with numerous spines, which are larger at the posterior margin and (iii) small, rounded tegumentary papillae around the opening of the oral sucker, which are morphologically different from those of the oral sucker itself, some of which are randomly disposed in the ventrolateral tegumentary region of the anterior third of the body. Addionally, based on SSU rDNA, a phylogenetic analysis including Brachycoeliidae and Mesocoeliidae taxa available on GenBank established the close relationship between M. lanfrediae sp. nov. and Mesocoelium sp.     

Focal adhesion kinase mediates cell survival via NF-kappaB and ERK signaling pathways

Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK^–/–) embryonic fibroblasts. FAK^–/– fibroblasts were more vulnerable to TNF--induced apoptosis, as measured by terminal deoxynucleotidyl transferase positivity. FAK^–/– fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK^+/+ fibroblasts. Insulin receptor substrate-1 expression was completely abolished and NF-B activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-x_L in FAK^–/– cells. Upon serum withdrawal, FAK^+/+ cells exhibited marked attenuation of basal ERK phosphorylation, while FAK^–/– cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased insulin receptor substrate (IRS)-2 expression in FAK^–/– cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-B and interaction between activated growth factor anti-apoptotic signaling pathways involving both phosphatidylinositol 3-kinase/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for upregulation of the anti-apoptotic NF-B response, as well as for normal expression of growth factor signaling proteins. Thus we propose a novel role for FAK in protection from cytokine-mediated apoptosis. apoptosis; ERK1/2; insulin; TNF-; IGF-1     

Survey of Vietnamese coffee beans for the presence of ochratoxigenic Aspergilli

Vietnamese coffee beans were investigated for the presence of ochratoxigenic Aspergilli. Ninety-three percent of the coffee samples studied were positive for A. niger. No other ochratoxigenic species were present. HPLC analysis determined that 8.7% of the A. niger strains were positive for ochratoxin A (OA) production. There was no significant difference in the level of contamination or incidence of toxigenic strains in samples that had been rejected by manual sorting and those that were destined for human consumption. No OA-producing fungi were uncovered in a fresh coffee bean sample analysed, suggesting that the OA problem most likely occurs post-harvest.     

sPLA2 and the epidermal barrier

The mammalian epidermis provides both an interface and a protective barrier between the organism and its environment. Lipid, processed into water-impermeable bilayers between the outermost layers of the epidermal cells, forms the major barrier that prevents water from exiting the organism, and also prevents toxins and infectious agents from entering. The secretory phospholipase 2 (sPLA2) enzymes control important processes in skin and other organs, including inflammation and differentiation. sPLA2 activity contributes to epidermal barrier formation and homeostasis by generating free fatty acids, which are required both for formation of lamellar membranes and also for acidification of the stratum corneum (SC). sPLA2 is especially important in controlling SC acidification and establishment of an optimum epidermal barrier during the first postnatal week. Several sPLA2 isoforms are present in the epidermis. We find that two of these isoforms, sPLA2 IIA and sPLA2 IIF, localize to the upper stratum granulosum and increase in response to experimental barrier perturbation. sPLA2F^−/− mice also demonstrate a more neutral SC pH than do their normal littermates, and their initial recovery from barrier perturbation is delayed. These findings confirm that sPLA2 enzymes perform important roles in epidermal development, and suggest that the sPLA2IIF isoform may be central to SC acidification and barrier function. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Syngeneic leukemia models using lentiviral transgenics

Animal models are necessary to study cancer and develop treatments. After decades of intensive research, effective treatments are available for only a few types of leukemia, while others are currently incurable. Our goal was to generate novel leukemia models in immunocompetent mice. We had achieved abilities for overexpression of multiple driving oncogenes simultaneously in normal primary cells, which can be transplanted and followed in vivo. Our experiments demonstrated the induction of primary malignant growth. Leukemia lines that model various types of leukemia, such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), were passaged robustly in congenic wild-type immunocompetent mice. These novel leukemia lines, which may complement previous models, offer the flexibility to generate tailored models of defined oncogenes of interest. The characterization of our leukemia models in immunocompetent animals can uncover the mechanisms of malignancy progression and offer a unique opportunity to stringently test anti-cancer chemotherapies.Subject terms: Cancer models, Haematopoietic stem cells, Leukaemia     

Activities of enzymes of acetoacetate metabolism in rat brown adipose tissue during development.

The activities of two mitochondrial enzymes concerned in the utilization of acetoacetate, namely 3-oxoacid CoA-transferase and acetoacetyl-CoA thiolase, were high throughout the suckling and weanling period in brown adipose tissue of the rat. In contrast, 3-hydroxybutyrate dehydrogenase activity was comparatively low during this period. The activity of cytosolic acetoacetyl-CoA synthetase (involved in lipogenesis) declined after birth and remained low until the pups were weaned. Experiments with brown-adipose-tissue slices from weanling rats indicated that 70% of the [3-14C]acetoacetate utilized was oxidized to 14CO2, and this value was not altered appreciably by the addition of glucose and insulin.     

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon.

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, approximately 80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V(1)) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K(171) and D(180) were observed in the matrix and one core protein with an amino-terminal sequence commencing at G(189) was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E(373)-A(374), E(1480)-G(1481) and E(1771)-A(1772). The analysis of versican catabolites (splice variants V(0) and/or V(1)) also showed evidence of degradation of the core protein by aggrecanase within the GAG-beta subdomain, as well as cleavage by other proteinase(s) within the GAG-alpha and GAG-beta subdomains of versican (variants V(0) and/or V(2)). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.     

Large aggregating and small leucine-rich proteoglycans are degraded by different pathways and at different rates in tendon.

This work investigated the kinetics of catabolism and the catabolic fate of the newly synthesized (35)S-labelled proteoglycans present in explant cultures of tendon. Tissue from the proximal region of bovine deep flexor tendon was incubated with [(35)S]sulfate for 6 h and then placed in explant cultures for periods of up to 15 days. The amount of radiolabel associated with proteoglycans and free [(35)S]sulfate lost to the medium and retained in the matrix was determined for each day in culture. It was shown that the rate of catabolism of radiolabelled small proteoglycans (decorin and biglycan) was significantly slower (T((1/2)) > 20 days) compared with the radiolabelled large proteoglycans (aggrecan and versican) that were rapidly lost from the tissue (T((1/2)) approximately 2 days). Both the small and large newly synthesized proteoglycans were lost from the matrix with either intact or proteolytically modified core proteins. When explant cultures of tendon were maintained either at 4 degrees C or in the presence of the lysosomotrophic agent ammonium chloride, inhibition of the cellular catabolic pathway for small proteoglycans was demonstrated indicating the involvement of cellular activity and lysosomes in the catabolism of small proteoglycans. It was estimated from these studies that approximately 60% of the radiolabelled small proteoglycans that were lost from the tissue were degraded by the intracellular pathway present in tendon cells. This work shows that the pathways of catabolism for large aggregating and small leucine-rich proteoglycans are different in tendon and this may reflect the roles that these two populations of proteoglycans play in the maintenance of the extracellular matrix of tendon.     

Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity.

Bovine joint capsule was maintained in explant culture in the presence of bovine aggrecan monomer and it was shown that the aggrecan monomer was degraded. Amino-terminal sequence analysis of the resulting aggrecan core protein fragments revealed that the core protein was cleaved at five specific sites attributed to glutamyl endopeptidases referred to as aggrecanase activity. Fibroblast cultures were established from explant cultures of joint capsule and when these cells were exposed to aggrecan, cleavage of the core protein of aggrecan at the aggrecanase sites was observed. Inclusion of either retinoic acid or interleukin-1alpha in medium of either joint capsule explant cultures or fibroblast cultures did not increase the rate of cleavage of exogenous aggrecan present in the culture medium. When aggrecan monomer was incubated with conditioned medium from explant cultures of joint capsule maintained in medium, degradation could be detected after 10 min. After a 6-h incubation period the same fragments of aggrecan core protein were observed as those for tissue or cells incubated directly with aggrecan monomer. RT-PCR analysis of mRNA extracted from joint capsule fibroblasts showed that these cells express both aggrecanase-1 and -2 [ADAMTS-2 (Tang) and ADAMTS-5].