Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.     

Temperature-pressure stability of green fluorescent protein: a Fourier transform infrared spectroscopy study

Green fluorescent protein (GFP) is widely used as a marker in molecular and cell biology. For its use in high-pressure microbiology experiments, its fluorescence under pressure was recently investigated. Changes in fluorescence with pressure were found. To find out whether these are related to structural changes, we investigated the pressure stability of wild-type GFP (wtGFP) and three of its red shift mutants (AFP, GFP(mut1), and GFP(mut2)) using Fourier transform infrared spectroscopy. For the wt GFP, GFP(mut1), and GFP(mut2) we found that up to 13-14 kbar the secondary structure remains intact, whereas AFP starts unfolding around 10 kbar. The 3-D structure is held responsible for this high-pressure stability. Previously observed changes in fluorescence at low pressure are rationalized in terms of the pressure-induced elastic effect. Above 6 kbar, loss of fluorescence is due to aggregation. Revisiting the temperature stability of GFP, we found that an intermediate state is populated along the unfolding pathway of wtGFP. At higher temperatures, the unfolding resulted in the formation of aggregates of wtGFP and its mutants.     

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 alpha (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre-hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.     

Recruitment Variability of Coral Reef Sessile Communities of the Far North Great Barrier Reef

One of the key components in assessing marine sessile organism demography is determining recruitment patterns to benthic habitats. An analysis of serially deployed recruitment tiles across depth (6 and 12 m), seasons (summer and winter) and space (meters to kilometres) was used to quantify recruitment assemblage structure (abundance and percent cover) of corals, sponges, ascidians, algae and other sessile organisms from the northern sector of the Great Barrier Reef (GBR). Polychaetes were most abundant on recruitment titles, reaching almost 50% of total recruitment, yet covered <5% of each tile. In contrast, mean abundances of sponges, ascidians, algae, and bryozoans combined was generally less than 20% of total recruitment, with percentage cover ranging between 15–30% per tile. Coral recruitment was very low, with <1 recruit per tile identified. A hierarchal analysis of variation over a range of spatial and temporal scales showed significant spatio-temporal variation in recruitment patterns, but the highest variability occurred at the lowest spatial scale examined (1 m—among tiles). Temporal variability in recruitment of both numbers of taxa and percentage cover was also evident across both summer and winter. Recruitment across depth varied for some taxonomic groups like algae, sponges and ascidians, with greatest differences in summer. This study presents some of the first data on benthic recruitment within the northern GBR and provides a greater understanding of population ecology for coral reefs.     

MyD88 plays a unique role in host defense but not arthritis development in Lyme disease

To assess the contribution of TLR signaling in the host response to Borrelia burgdorferi, mice deficient in the common TLR adaptor protein, myeloid differentiation factor 88 (MyD88), were infected with B. burgdorferi. MyD88-deficient mice harbored extremely high levels of B. burgdorferi in tissues when compared with wild-type littermates and greater amounts of spirochetes in tissues than TLR2-deficient mice. These findings suggest that, in addition to TLR2, other MyD88-dependent pathways play a significant role in the host defense to B. burgdorferi. MyD88(-/-) mice maintained the ability to produce Abs directed against B. burgdorferi. Partial clearance of spirochetes was evident in long term infection studies and immune sera from MyD88-deficient mice were able to protect naive mice from infection with B. burgdorferi. Thus, the acquired immune response appeared to be functional in MyD88(-/-) mice, and the inability to control spirochete numbers was due to a failure of cells involved in innate defenses. Although macrophages from MyD88(-/-) mice responded poorly to Borrelia sonicate in vitro, MyD88(-/-) mice still developed an inflammatory arthritis after infection with B. burgdorferi characterized by an influx of neutrophils and mononuclear cells. The findings presented here point to a dichotomy between the recruitment of inflammatory cells to tissue and an inability of these cells to kill localized spirochetes.     

The probability and chromosomal extent of trans-specific polymorphism

Balancing selection may result in trans-specific polymorphism: the maintenance of allelic classes that transcend species boundaries by virtue of being more ancient than the species themselves. At the selected site, gene genealogies are expected not to reflect the species tree. Because of linkage, the same will be true for part of the surrounding chromosomal region. Here we obtain various approximations for the distribution of the length of this region and discuss the practical implications of our results. Our main finding is that the trans-specific region surrounding a single-locus balanced polymorphism is expected to be quite short, probably too short to be readily detectable. Thus lack of obvious trans-specific polymorphism should not be taken as evidence against balancing selection. When trans-specific polymorphism is obvious, on the other hand, it may be reasonable to argue that selection must be acting on multiple sites or that recombination is suppressed in the surrounding region.     

The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products

The beta-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of beta-1,4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of beta-1,4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (betaalpha)(8)-barrel architecture. However, BLGAL betaalpha-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long betaalpha-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL.     

Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D¹²⁷xxxLL^131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.     

Endogenous expression of liver-specific drug transporters for organic anions in the rat hepatocytoma fusion cell line HPCT-1E3

HPCT-1E3 cells, a fusion cell line between primary rat hepatocytes and Fao Reuber hepatoma cells H35, are immortalized hybrid cells with many phenotypic properties of liver parenchyma including phase I and II metabolism and bile acid secretion. Selective elimination of endogenous compounds and drugs by the liver involves transport proteins that complementarily mediate uptake and efflux in co-operation with metabolism, but the study of this function is limited by the unavailability of an integrated in vitro model. Therefore, we investigated the expression of some important liver-specific import and export carrier proteins for organic anions in this cell line. RT-PCR analysis indicated gene expression of Oat2, Oatplal, Oatpla4, Oatplb2, Rfc-1/MTX-1, FOLR, Mrp1-6, mdr1, and Lrp. Uptake and efflux as well as inhibition studies confirmed the functional activity of Oat, Oatp, Rfc-1, Mrp, and Mdr carriers. In conclusion, the hepatocyte-like HPCT-1E3 cell line shows endogenous expression of all liver-specific carrier proteins for organic anions and may hence represent a valuable in vitro model for the study of transport phenomena and their regulation in hepatocytes.     

Phenotype–Genotype Analysis of Chinese Patients with Early-Onset LMNA-Related Muscular Dystrophy

This study aimed to analyze the correlation between the phenotype and genotype of Chinese patients with early-onset lamin A (LMNA)-related muscular dystrophy (MD). The clinical and myopathological data of 21 Chinese pediatric patients with early-onset LMNA-related MD were collected and analyzed. LMNA gene mutation analysis was performed by direct sequencing of genomic DNA. Sublocalization of wild-type and mutant proteins were observed by immunofluorescence using cultured fibroblasts and human embryonic kidney 293 (HEK 293) cell. Seven patients were diagnosed with Emery-Dreifuss muscular dystrophy (EDMD) and 14 were diagnosed with LMNA-associated congenital muscular dystrophy (L-CMD). Four biopsy specimens from the L-CMD cases exhibited inflammatory changes. Abnormal nuclear morphology was observed with both transmission electron microscopy and lamin A/C staining. We identified 10 novel and nine known LMNA gene mutations in the 21 patients. Some mutations (c.91G>A, c.94_96delAAG, c.116A>G, c.745C>T, c.746G>A, and c.1580G>C) were well correlated with EDMD or L-CMD. LMNA-related MD has a common symptom triad of muscle weakness, joint contractures, and cardiac involvement, but the severity of symptoms and disease progression differ greatly. Inflammatory change in biopsied muscle is a characteristic of early-stage L-CMD. Phenotype–genotype analysis determines that some mutations are well correlated with LMNA-related MD.