Physical properties and biological interactions of liposomes developed as a drug carrier in the field of regenerative medicine

Liposomes are used for encapsulation of the active compounds in different therapies, with the increasing frequency. The important areas of clinical applications of liposomes are cancer targeted treatment, antibiotic delivery or regenerative medicine. The liposomes can transfer both hydrophilic and hydrophobic compounds and have the lipid bilayer which imitates the cell membrane. Liposomes additionally may extend half-live period of drugs and protect them against the elimination in different ways, such as phagocytosis, enzymatic cleavage or exclusion by detoxification. The size and charge of liposomes play an important role in drug distribution and absorption into the cell. Limited data is available on the effects of liposomes on stem cells and progenitor cells. In this article, we examined the effect of charged conventional liposomes on growth of mesenchymal and blood stem cells isolated from umbilical cord. The data suggest a likelihood, that positively charged liposomes could impair stem cell growth and metabolism. Different methodological approaches allowed for the selection of negatively charged liposomes for further experiments, as the only type of liposomes which has the lowest cytotoxicity and does not affect hematopoietic cell proliferation.     

Differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics

alpha-Dystroglycan (alpha-DG) was recently identified as a receptor for lymphocytic choriomeningitis virus (LCMV) and several other arenaviruses, including Lassa fever virus (W. Cao, M. D. Henry, P. Borrow, H. Yamada, J. H. Elder, E. V. Ravkov, S. T. Nichol, R. W. Compans, K. P. Campbell, and M. B. A. Oldstone, Science 282:2079-2081, 1998). Data presented in this paper indicate that the affinity of binding of LCMV to alpha-DG determines viral tropism and the outcome of infection in mice. To characterize this relationship, we evaluated the interaction between alpha-DG and several LCMV strains, variants, and reassortants. These viruses could be divided into two groups with respect to affinity of binding to alpha-DG, dependence on this protein for cell entry, viral tropism, and disease course. Viruses that exhibited high-affinity binding to alpha-DG displayed a marked dependence on alpha-DG for cell entry and were blocked from infecting mouse 3T6 fibroblasts by 1 to 4 nM soluble alpha-DG. In addition, high-affinity binding to alpha-DG correlated with an ability to infiltrate the white pulp (T-dependent) area of the spleen, cause ablation of the cytotoxic T-lymphocyte (CTL) response by day 7 postinfection, and establish a persistent infection. In contrast, viruses with a lower affinity of binding to alpha-DG were only partially inhibited from infecting alpha-DG(-/-) embryonic stem cells and required a concentration of soluble alpha-DG higher than 100 nM to prevent infection of mouse 3T6 fibroblasts. These viruses that bound at low affinity were mainly restricted to the splenic red pulp, and the host generated an effective CTL response that rapidly cleared the infection. Reassortants of viruses that bound to alpha-DG at high and low affinities were used to map genes responsible for the differences described to the S RNA, containing the virus attachment protein glycoprotein 1.     

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.     

Structural and functional identification of major histocompatibility complex class I-restricted self-peptides as naturally occurring molecular mimics of viral antigens. Possible role in CD8+ T cell-mediated, virus-induced autoimmune disease

Structural similarity (molecular mimicry) between viral epitopes and self-peptides can lead to the induction of autoaggressive CD4(+) as well as CD8(+) T cell responses. Based on the flexibility of T cell receptor/antigen/major histocompatibility complex recognition, it has been proposed that a self-peptide could replace a viral epitope for T cell recognition and therefore participate in pathophysiological processes in which T cells are involved. To address this issue, we used, as a molecular model of viral antigen, the H-2D(b)-restricted immunodominant epitope nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic choriomeningitis virus (LCMV). We identified peptide sequences from murine self-proteins that share structural and functional homology with LCMV NP-(396-404) and that bound to H-2D(b) with high affinity. One of these self-peptides, derived from tumor necrosis factor receptor I (FGPSNWHFM, amino acids 302-310), maintained LCMV-specific CD8(+) T cells in an active state as observed both in vitro in cytotoxic assays and in vivo in a model of virus-induced autoimmune diabetes, the rat insulin promoter-LCMV NP transgenic mouse. The natural occurrence and molecular concentration at the surface of H-2(b) spleen cells of tumor necrosis factor receptor I-(302-310) were determined by on-line micro-high pressure liquid chromatography/mass spectrometry and supported its biological relevance.     

A global S. cerevisiae small ubiquitin‐related modifier (SUMO) system interactome

The small ubiquitin‐related modifier (SUMO) system has been implicated in a number of biological functions, yet the individual components of the SUMO machinery involved in each of these activities were largely unknown. Here we report the first global SUMO system interactome. Using affinity purification coupled with mass spectrometry, we identify >450 protein–protein interactions surrounding the SUMO E2, Siz type E3s and SUMO‐specific proteases in budding yeast. Exploiting this information‐rich resource, we validate several Siz1‐ and Siz2‐specific substrates, identify a nucleoporin required for proper Ulp1 localization, and uncover important new roles for Ubc9 and Ulp2 in the maintenance of ribosomal DNA.     

Mass spectrometry reveals specific and global molecular transformations during viral infection

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.     

Lymphotoxin-alpha- and lymphotoxin-beta-deficient mice differ in susceptibility to scrapie: evidence against dendritic cell involvement in neuroinvasion

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders of humans and animals often initiated by oral intake of an infectious agent. Current evidence suggests that infection occurs initially in the lymphoid tissues and subsequently in the central nervous system (CNS). The identity of infected lymphoid cells remains controversial, but recent studies point to the involvement of both follicular dendritic cells (FDC) and CD11c(+) lymphoid dendritic cells. FDC generation and maintenance in germinal centers is dependent on lymphotoxin alpha (LT-alpha) and LT-beta signaling components. We report here that by the oral route, LT-alpha -/- mice developed scrapie while LT-beta -/- mice did not. Furthermore, LT-alpha -/- mice had a higher incidence and shorter incubation period for developing disease following inoculation than did LT-beta -/- mice. Transplantation of lymphoid tissues from LT-beta -/- mice, which have cervical and mesenteric lymph nodes, into LT-alpha -/- mice, which do not, did not alter the incidence of CNS scrapie. In other studies, a virus that is tropic for and alters functions of CD11c(+) cells did not alter the kinetics of neuroinvasion of scrapie. Our results suggest that neither FDC nor CD11c(+) cells are essential for neuroinvasion after high doses of RML scrapie. Further, it is possible that an as yet unidentified cell found more abundantly in LT-alpha -/- than in LT-beta -/- mice may assist in the amplification of scrapie infection in the periphery and favor susceptibility to CNS disease following peripheral routes of infection.     

Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells

A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl-tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl-tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells.     

Cloning, sequence analysis, and processing of the rat and human atrial natriuretic peptide precursors

Complementary DNA sequences and structural genes encoding the atrial natriuretic peptide precursor (prepro-ANP) have been cloned. Analysis of DNA sequences, complementary to rat atrial prepro-ANP mRNA, has revealed that the various natriuretic peptides isolated from rat atrium reside at the carboxy terminus of a 152-amino-acid precursor protein. The human gene, comprised of three exons and two intervening sequences, encodes a protein of 151 amino acids highly homologous to the rat precursor. Although putative proteolytic processing sites can be identified throughout the prepro-ANP amino acid sequence, the natural form of the mature ANP has not been identified. Therefore, the sites and mechanisms of prepro-ANP processing to mature peptides forms are unknown. However, the successful cloning of the prepro-ANP gene and corresponding cDNAs provide the necessary molecular tools to address these fundamental questions relating to the regulation of ANP synthesis and processing in atrial and extraatrial tissues.