Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers

Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) - the major component of pulmonary surfactant - using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (T(m)) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on T(m) and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS)>K(PFOA)>K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds cause adverse biological effects.     

Iron status and oxidative stress in beta-thalassemia patients in Jakarta

A study on thalassemia intermedia and major patients in Jakarta was initiated to obtain a comprehensive picture of metabolic dysregulation, iron overload, oxidative stress, and cell damage. Data are presented from a group of 14 transfusion-dependent patients in an age range of 11-25 years (T) and another group of 9 frequently transfused (for at least 15 years) patients aged 17-30 years (L). A third group comprised 6 patients (aged 7 to 14 years) who had not yet obtained transfusions (N). The 21 controls (C) were voluntary students without diagnosis or clinical signs of thalassemia up to 30 years of age. The study was approved by the Ethical Clearance Board of the Medical Faculty and all blood samples from controls and patients were obtained on fully informed consent. Levels of antioxidants (vitamins A, C, E and beta-carotene) and reactive thiols are considerably decreased in transfused patients, whereas signs of iron overload and cell damage are increased (serum iron, ferritin, transferrin saturation, SGOT, SGPT, gamma-GT, bilirubin). Results can be summarized that non-transfused thalassemia intermedia patients exert slight signs of oxidative stress, and increased hemoglobin degradation but no significant indication of tissue or cell damage. This picture differs considerably from transfusion-dependent thalassemia major patients: highly significant decrease in antioxidants and thiols and tremendous iron overload and cell damage. The picture is even worsened in long-term transfused patients. Iron chelation after transfusion is not sufficient in Indonesia, because it is normally (with few exceptions) applied only once together with transfusion. Hence, one major reason of the bad condition of transfusion-dependent thalassemia patients in Indonesia appears to be frequent transfusions (on the average one per month) and insufficient chelation of one treatment per month together with transfusion.     

Mechanism of the maturation process of SARS-CoV 3CL protease

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.     

Black lipid membranes of tetraether lipids from Thermoplasma acidophilum.

Black lipid membranes were formed of tetraether lipids from Thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. Bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. Measurements of the specific membrane capacitance (Cm = 0.744 microF/cm2) showed that the membrane-spanning tetraether lipids from Thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 nm no matter from which solvent. This finding corresponds to the results of Gliozzi et al. for the lipids of another archaebacterium, Sulfolobus solfataricus. Black lipid membranes were formed at room temperature with a torus from bilayer-forming lipids, however, the torus could also be formed by the tetraether-lipid itself at room temperature and at defined concentration. In these stable black lipid membranes, conductance was measured in the presence of valinomycin, nonactin, and gramicidin. At 10(-7) M concentration, valinomycin mediated higher conductance in membranes from tetraether lipids (200-1200 microS/cm2) than from bilayer-forming lipids (125-480 microS/cm2). Nonactin, at 10(-6) M concentration, mediated a 6-fold higher conductance in a tetraether lipid membrane than in a bilayer, whereas conductance, in the presence of 5 x 10(-11) M gramicidin could reach higher values in bilayers than in tetraether lipid monolayers of comparable thickness. Monensin did not increase the conductance of black lipid membranes from tetraether lipids under all conditions applied in our experiments. Poly(L-lysine) destroyed black lipid membranes. Lipopolysaccharides from Thermoplasma acidophilum were not able to form stable black lipid membranes by themselves. The lipopolysaccharide complexes from Thermoplasma acidophilum and from Escherichia coli decreased the valinomycin-mediated conductance of monolayer and bilayer membranes. This influence was stronger than that of the polysaccharide dextran.     

Protease-activated receptors in the musculoskeletal system

Protease-activated receptors (PARs) mediate cellular responses to a subset of extracellular proteases, including blood coagulation factors and proteases produced by inflammatory cells. Cells in bone, cartilage and muscle exhibit cell type-specific expression patterns and functional responses for the different PARs. Activators of PAR-1 include thrombin, and activators of PAR-2 include trypsin and tryptase; PARs-3 and -4 are also receptors for thrombin. Thrombin stimulates PAR-1-mediated proliferative responses in osteoblasts, chondrocytes and myoblasts, and in developing muscle, PAR-1 activation by thrombin appears to mediate activity-dependent polyneuronal synapse reduction. In bone, activation of PAR-2 leads to inhibition of osteoblast-mediated osteoclast differentiation induced by hormones or cytokines, and in muscle, PAR-2 activation leads to stimulation of myoblast proliferation. Although there is some evidence for a role for PARs expressed by cells of the musculoskeletal system at specific stages of development, their major role appears to be in protecting the tissues from the destructive effects of inflammation and promoting regeneration. This review discusses the regulation of cell function in the musculoskeletal system by receptor-mediated responses to proteases. Expression patterns of PARs, the circumstances in which PAR activators are likely to be present, functional responses of PAR activation, and responses to thrombin for which receptors have not yet been identified are considered.     

Genomic diversity and differentiation of a managed island wild boar population

The evolution of island populations in natural systems is driven by local adaptation and genetic drift. However, evolutionary pathways may be altered by humans in several ways. The wild boar (WB) (Sus scrofa) is an iconic game species occurring in several islands, where it has been strongly managed since prehistoric times. We examined genomic diversity at 49 803 single-nucleotide polymorphisms in 99 Sardinian WBs and compared them with 196 wild specimens from mainland Europe and 105 domestic pigs (DP; 11 breeds). High levels of genetic variation were observed in Sardinia (80.9% of the total number of polymorphisms), which can be only in part associated to recent genetic introgression. Both Principal Component Analysis and Bayesian clustering approach revealed that the Sardinian WB population is highly differentiated from the other European populations (F_ST=0.126–0.138), and from DP (F_ST=0.169). Such evidences were mostly unaffected by an uneven sample size, although clustering results in reference populations changed when the number of individuals was standardized. Runs of homozygosity (ROHs) pattern and distribution in Sardinian WB are consistent with a past expansion following a bottleneck (small ROHs) and recent population substructuring (highly homozygous individuals). The observed effect of a non-random selection of Sardinian individuals on diversity, F_ST and ROH estimates, stressed the importance of sampling design in the study of structured or introgressed populations. Our results support the heterogeneity and distinctiveness of the Sardinian population and prompt further investigations on its origins and conservation status.     

Right or left turn? RecA family protein filaments promote homologous recombination through clockwise axial rotation

The RecA family proteins mediate homologous recombination, a ubiquitous mechanism for repairing DNA double-strand breaks (DSBs) and stalled replication forks. Members of this family include bacterial RecA, archaeal RadA and Rad51, and eukaryotic Rad51 and Dmc1. These proteins bind to single-stranded DNA at a DSB site to form a presynaptic nucleoprotein filament, align this presynaptic filament with homologous sequences in another double-stranded DNA segment, promote DNA strand exchange and then dissociate. It was generally accepted that RecA family proteins function throughout their catalytic cycles as right-handed helical filaments with six protomers per helical turn. However, we recently reported that archaeal RadA proteins can also form an extended right-handed filament with three monomers per helical turn and a left-handed protein filament with four monomers per helical turn. Subsequent structural and functional analyses suggest that RecA family protein filaments, similar to the F1-ATPase rotary motor, perform ATP-dependent clockwise axial rotation during their catalytic cycles. This new hypothesis has opened a new avenue for understanding the molecular mechanism of RecA family proteins in homologous recombination.     

Canine embryonic stem cells: State of the art

Embryonic stem cells (ESCs) are permanent cell lines that can be maintained in a pluripotent, undifferentiated state. Appropriate environmental stimuli can cause them to differentiate into cell types of all three germ layers both in vitro and in vivo. Embryonic stem cells bear many opportunities for clinical applications in tissue engineering and regenerative medicine. Whereas most of our knowledge on the biology and technology of ESCs is derived from studies with mouse cells, large animal models mimicking important aspects of human anatomy, physiology, and pathology more closely than mouse models are urgently needed for studies evaluating the safety and efficacy of cell therapies. The dog is an excellent model for studying human diseases, and the availability of canine ESCs would open new possibilities for this model in biomedical research. In addition, canine ESCs could be useful for the development of cell-based approaches for the treatment of dogs. Here, we discuss the features of recently reported canine embryo-derived cells and their potential applications in basic and translational biomedical research.     

Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.