A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Inhibitors of the tissue factor/factor VIIa-induced coagulation: synthesis and in vitro evaluation of novel 2-aryl substituted pyrid

The synthesis of a series of 2-aryl substituted hetero annulated 1,3-oxazin-4-ones and their evaluation as specific inhibitors of the tissue factor (TF)/factor VIIa (FVIIa)-induced pathway of coagulation is reported. Inhibitory activities (IC₅₀ values) in the range 0.64 to >40 μM on the activation of factor X (FX) by the TF/FVIIa complex were found for compounds having one or two electronegative substituents such as F and NO₂ in the 2-aryl substituent. Some of the compounds showed a selectivity ratio towards FX and thrombin of >50, thus being similar in specificity to 2-aryl substituted 4H-3,1-benzoxazin-4-ones described as potential drugs for oral antithrombotic treatment without side effects, such as bleeding, which is observed especially with thrombin inhibitors.     

Glandular kallikreins of the cotton-top tamarin: molecular cloning of the gene encoding the tissue kallikrein

The glandular kallikrein family is composed of structurally related serine proteases. Studies show that the mouse family encompasses at least 14 highly conserved functional genes, but of these only the tissue kallikarein has a human ortholog. In man, the tissue kallikrein display high sequence similarity with prostate specific antigen and human glandular kallikrein 2, suggesting that they evolved after the separation of primates and rodents. A phylogenetic study of the genes encoding glandular kallikreins in species evolutionarily located between rodents and man may reveal interesting details on how the gene family evolved, which in turn could yield information about the function of the proteins. Therefore, we have initiated a study of the glandular kallikreins of the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report the cloning and nucleotide sequence of one of these, the tissue kallikrein gene. The gene of 4.4 kb is composed of five exons, and the structure is 90% similar to that of the orthologous human gene. It gives rise to a polypeptide of 261 amino acids, including a signal peptide of 17 residues, a pro-piece of 7 residues, and the mature protein of 237 residues with an estimated molecular mass of 26.3 kD. The similarity to the human prostate specific antigen and human glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and flanking regions. The lower similarity to these genes compared with the human tissue kallikrein gene indicates that they, or a progenitor to them, arose in primates prior to the separation of New and Old World monkeys. Genomic Southern blots also show that the cotton-top tamarin genome encompasses at least one more glandular kallikrein gene.     

Two systems for phosphate uptake in Yarrowia lipolytica cells grown at acidic conditions

Inorganic phosphate (Pi) is accumulated by Yarrowia lipolytica cells grown at acidic pH conditions by two kinetically discrete H+/Pi-cotransport systems with apparent K(m) values for Pi of 12-18 microM and 2-3 mM Pi at pH 5.5, respectively. One of these is derepressible and operates at low external Pi concentrations; the other is most likely constitutively expressed and comes into play at high Pi concentrations. The derepression of the high-affinity Pi transport system is under the control of available extracellular Pi as well as the amount of intracellular polyphosphates stores. Characteristics of the Pi transport behavior in Yarrowia lipolytica are discussed.     

Transition state analysis of the complex between coagulation factor VIIa and tissue factor: suggesting a sequential domain-binding pathway

Injury of a blood vessel exposes membrane-bound tissue factor (TF) to blood, which allows binding of coagulation factor VIIa (FVIIa). This initiation of the coagulation cascade is dictated by a specific multi-domain interaction between FVIIa and TF. To examine the energies involved in the transition state of the FVIIa:TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with a smaller cysteine residue. Determination of Phi values in each of the positions using surface plasmon resonance measurements enabled us to characterize the transition state complex between the resulting sTF variants and FVIIa. We found that the interactions in the transition state seemed to be most pronounced between the protease domain of FVIIa and sTF while detailed specific interactions between the Gla-domain and sTF were missing. Thus, the transition state energy data indicate a sequential binding event between these two macromolecules.     

2B4/CD48-mediated regulation of lymphocyte activation and function

2B4 (CD244) is a member of the CD2 subset of the Ig superfamily. This molecule is expressed on innate immune cells, including NK cells, and on subsets of T cells. The 2B4 molecule interacts with CD48, which is widely expressed on hemopoietic cells. Although earlier reports demonstrated a role for 2B4 as an activating receptor in both mice and humans, recent studies of 2B4-deficient mice have suggested that 2B4 functions predominantly as an inhibitory receptor in mice. In addition, 2B4 may also act as a costimulatory ligand for cells expressing CD48. Thus, the 2B4 molecule is more multifunctional than previously understood. In this study, we delineate the current view of 2B4-CD48 interactions among lymphocytes and other cells.     

915 MHz microwaves and 50 Hz magnetic field affect chromatin conformation and 53BP1 foci in human lymphocytes from hypersensitive and healthy persons

We used exposure to microwaves from a global system for mobile communication (GSM) mobile phone (915 MHz, specific absorption rate (SAR) 37 mW/kg) and power frequency magnetic field (50 Hz, 15 muT peak value) to investigate the response of lymphocytes from healthy subjects and from persons reporting hypersensitivity to electromagnetic field (EMF). The hypersensitive and healthy donors were matched by gender and age and the data were analyzed blind to treatment condition. The changes in chromatin conformation were measured with the method of anomalous viscosity time dependencies (AVTD). 53BP1 protein, which has been shown to colocalize in foci with DNA double strand breaks (DSBs), was analyzed by immunostaining in situ. Exposure at room temperature to either 915 MHz or 50 Hz resulted in significant condensation of chromatin, shown as AVTD changes, which was similar to the effect of heat shock at 41 degrees C. No significant differences in responses between normal and hypersensitive subjects were detected. Neither 915 MHz nor 50 Hz exposure induced 53BP1 foci. On the contrary, a distinct decrease in background level of 53BP1 signaling was observed upon these exposures as well as after heat shock treatments. This decrease correlated with the AVTD data and may indicate decrease in accessibility of 53BP1 to antibodies because of stress-induced chromatin condensation. Apoptosis was determined by morphological changes and by apoptotic fragmentation of DNA as analyzed by pulsed-field gel electrophoresis (PFGE). No apoptosis was induced by exposure to 50 Hz and 915 MHz microwaves. In conclusion, 50 Hz magnetic field and 915 MHz microwaves under specified conditions of exposure induced comparable responses in lymphocytes from healthy and hypersensitive donors that were similar but not identical to stress response induced by heat shock.     

Proteomics of Synechocystis sp. strain PCC 6803: identification of plasma membrane proteins

Cyanobacteria are unique prokaryotes since they in addition to outer and plasma membranes contain the photosynthetic membranes (thylakoids). The plasma membranes of Synechocystis 6803, which can be completely purified by density centrifugation and polymer two-phase partitioning, have been found to be more complex than previously anticipated, i.e. they appear to be essential for assembly of the two photosystems. A proteomic approach for the characterization of cyanobacterial plasma membranes using two-dimensional gel electrophoresis and mass spectrometry analysis revealed a total of 57 different membrane proteins of which 17 are integral membrane spanning proteins. Among the 40 peripheral proteins 20 are located on the periplasmic side of the membrane, while 20 are on the cytoplasmic side. Among the proteins identified are subunits of the two photosystems as well as Vipp1, which has been suggested to be involved in vesicular transport between plasma and thylakoid membranes and is thus relevant to the possibility that plasma membranes are the initial site for photosystem biogenesis. Four subunits of the Pilus complex responsible for cell motility were also identified as well as several subunits of the TolC and TonB transport systems. Several periplasmic and ATP-binding proteins of ATP-binding cassette transporters were also identified as were two subunits of the F(0) membrane part of the ATP synthase.     

Thyroid gland function in ovariectomized ewes exposed to phytoestrogens

Phytoestrogens are by definition plant-derived substances that are able to activate the mammalian oestrogen receptors. We examined the possible effects of phytoestrogens on the secretion of thyroid hormones as well as on the immunoreactivity to oestrogen receptor alpha (ER alpha) in the thyroid glands of ovariectomized ewes. Eight ovariectomized ewes were fed 3.5 kg of 100% red clover silage for 14 days. Blood samples were collected before and on day 14 of exposure to phytoestrogens. After 5 months, four of the ewes were re-exposed to red clover silage as described above and the other four served as controls. Blood samples were collected as above. All ewes were slaughtered at the end of the experiment and the thyroid glands were weighed and examined for macroscopical changes. Tissue samples were taken for immunohistochemistry and image analysis. Ewes exposed to red clover silage had significantly higher plasma concentrations of total T(3) and free T(3) than ewes fed hay. The cross-section area of thyroid follicles tended to be larger in ewes fed red clover silage than in the control animals. ER alpha immunoreactivity was stronger in thyroid glands from ewes exposed to phytoestrogens than in ewes fed hay. In conclusion, daily ingestion of 81-95 mg phytoestrogens per kg body weight for 14 days stimulated secretion of thyroid hormones and tended to increase follicle size and ER alpha immunoreactivity of thyroid glands of ovariectomized ewes.