HPTLC chromatography of androstene derivates. Application of normal phase thin-layer chromatographic retention data in QSAR studies

The chromatographic behavior of seven 16-oximino derivatives of 3beta-hydropxy-5-androstene have been investigated using the normal-phase (NP) HPTLC chromatographic mode of the type silica-non-polar diluent (benzene)-polar modifier (acetonitrile, ethyl acetate, or dioxane). The linear relationship between the retention constants (R(M)) and the logarithm of the organic modifier content in the mobile phase allowed for the calculation of R(M)0 values. The influence of substituent in the molecule on extrapolated retention data is discussed. To better understand the retention mechanism in the separation of androstene compounds, the functional group contributions (tauX) were compared with Hansch substituent constants (pi). An attempt to quantitate the lipophilicity of the investigated compounds using normal phase thin-layer chromatographic R(M)0 value was made. Also, the relative lipophilicity values determined previously by RPC as well as activity were compared with NPC data.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Functional imaging of pericellular proteolysis in cancer cell invasion

Proteolytic interactions between cells and extracellular matrix (ECM) are involved in many physiological and pathological processes, such as embryogenesis, wound healing, immune response, and cancer. The visualization of cell-mediated proteolysis towards ECM is thus required to understand basic mechanisms of tissue formation and repair, such as the breakdown and structural remodelling of ECM, inflammatory changes of tissue integrity, and the formation of proteolytic trails by moving cells. A panel of synergistic techniques for the visualization of pericellular proteolysis in live and fixed samples allow monitoring the of proteolytic tumor cell invasion in three-dimensional (3D) fibrillar collagen matrices in vitro. These include the quantification of collagenolysis by measuring the release of collagen fragments, the detection of protease expression and local activity by dequenching of fluorogenic substrate, and the staining of cleavage-associated neoepitopes together with changes in matrix structure. In combination, these approaches allow the high-resolution mapping of pericellular proteolysis towards ECM substrata including individual focal cleavage sites and the interplay between cell dynamics and alterations in the tissue architecture.     

Sex-specific effect of insulin-dependent diabetes 4 on regulation of diabetes pathogenesis in the nonobese diabetic mouse

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD x NOR) x NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.     

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class II (MHC II) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC II chaperone Ii, and hence in the formation of MHC II-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, Ii processing and MHC II peptide loading proceeded similarly in all three DC populations. We then analyzed MHC II localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.     

Polymerization of mycobacterial arabinogalactan and ligation to peptidoglycan

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(alpha1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf)1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5- and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[14C]Galp and recombinant UDP-Galp mutase as the source of [14C]Galf for galactan biosynthesis and 5-P-[14C]ribosyl-P-P as a donor of [14C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf)n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LU-arabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.     

Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn(2+)) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn(2+) complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO(4) or MnCl(2). In response to a pH gradient or a Mn(2+) ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn(2+) suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn(2+) complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn(2+) complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

Molecular dynamics and IR spectroscopy in investigation of phase transitions in molecular crystal 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl

Molecular dynamics (MD) simulations combined with temperature-dependent IR spectroscopic measurements were used to study phase transitions in molecular crystals of the mesogenic diol 4,4'-bis(11-hydroxy-1-undecyloxy)biphenyl. DSC measurements revealed four phase transitions in this molecular crystal at approximately 327.1 K, 389.8 K, 419.1 K and 431.9 K. Analysis of the dynamic trajectories at temperatures of 300 K, 360 K, 400 K and 480 K revealed changes in conformation of the mesogenic diol molecules and consequently changes in crystal packing and crystal structure in the temperature range 300-480 K and enabled us to understand the mechanism of the phase transitions.     

Proteolytic and non-proteolytic migration of tumour cells and leucocytes

The migration of different cell types, such as leucocytes and tumour cells, involves cellular strategies to overcome the physical resistance of three-dimensional tissue networks, including proteolytic degradation of extracellular matrix (ECM) components. High-resolution live-cell imaging techniques have recently provided structural and biochemical insight into the differential use of matrix-degrading enzymes in the migration processes of different cell types within the three-dimensional ECM. Proteolytic migration is achieved by slow-moving cells, such as fibroblasts and mesenchymally moving tumour cells, by engaging matrix metalloproteinases, cathepsins and serine proteases at the cell surface in a focalized manner ('pericellular proteolysis'), while adhesion and migratory traction are provided by integrins. Pericellular breakdown of ECM components generates localized matrix defects and remodelling along migration tracks. In contrast with tumour cells, constitutive non-proteolytic migration is used by rapidly moving T lymphocytes. This migration type does not generate proteolytic matrix remodelling, but rather depends on shape change to allow cells to glide and squeeze through gaps and trails present in connective tissues. In addition, constitutive proteolytic migration can be converted into non-proteolytic movement by protease inhibitors. After the simultaneous inhibition of matrix metalloproteinases, serine/threonine proteases and cysteine proteases in tumour cells undergoing proteolysis-dependent movement, a fundamental adaptation towards amoeboid movement is able to sustain non-proteolytic migration in these tumour cells (the mesenchymal-amoeboid transition). Instead of using proteases for matrix degradation, the tumour cells use leucoyte-like strategies of shape change and squeezing through matrix gaps along tissue scaffolds. The diversity of protease function in cell migration by different cell types highlights response diversity and molecular adaptation of cell migration upon pharmacotherapeutic protease inhibitor treatment.