Thermodynamic and kinetic studies on the mechanism of binding of methylumbelliferyl galactosides to the basic agglutinin from winged bean (Psophocarpus tetragonolobus)

The binding of winged bean basic agglutinin (WBA I) to 4-methylumbelliferyl (MeUmb) galactosides was examined by extrinsic fluorescence titration and stopped-flow spectrofluorimetry. Upon binding to WBA I, MeUmb alpha-galactosides show quenching in fluorescence intensity, decrease in UV absorbance with a concomitant blue shift, and decrease in fluorescence excited-state lifetimes. However, their beta-analogues show enhancement in fluorescence intensity, increase in UV absorbance with a red shift, and an increase in fluorescence excited-state lifetimes. This implies that the umbelliferyl groups of alpha- and beta-galactosides experience non-polar and polar microenvironments, respectively, upon binding to WBA I. Replacement of the anomeric hydroxyl group of galactose by 4-methylumbelliferyl moiety increases the affinity of resulting saccharides. Substitution of C-2 hydroxyl of galactose by an acetamido group leads to increased affinity due to a favorable entropy change. This suggests that acetamido group of MeUmb-alpha/beta-GalNAc binds to a relatively non-polar subsite of WBA I. Most interestingly, this substitution also reduces the association rate constants dramatically. Inspection of the activation parameters reveals that the enthalpy of activation is the limiting factor for the differences in the forward rate constants for these saccharides and the entropic contribution to the activation energy is small

Crystallization and Preliminary X-ray Studies of the Basic Lectin from Winged Bean (Psophocarpus tetragonolobus)

The basic lectin from winged bean (Psophocarpus tetragonolobus) could be crystallized using polyethyleneglycol (PEG) 4000 (I), PEG 8000 (II) and 2-methylpentane-2,4-diol (MPD) (III) as precipitants. Crystal forms I and II grew in the presence of methyl-α-Image -galactopyranoside or N -acetylgalactosamine while III grew in the absence of sugar. The three forms have the same space group (P21212) and similar unit cell dimensions with two dimeric molecules in the asymmetric unit. The unit cell dimensions are a = 156·8 Å, b = 89·0 Å, c = 73·3 Å for I, a = 155·5 Å, b = 92·3 Å, c = 72·5 Å for II and a = 148·3 Å, b = 90·7 Å, c = 73·8 Å for III. The crystals, particularly those grown using PEG 8000, are suitable for high resolution X-ray analysis, which is in progress.

Thermodynamics of the binding of galactopyranoside derivatives to the basic lectin from winged bean (Psophocarpus tetrogonolobus)

The thermodynamics of the binding of D-galactopyranoside (Gal), 2-acetamido-2-deoxygalactopyranoside (GalNAc), methyl-alpha-D-galactopyranoside, and methyl-beta-D-galactopyranoside to the basic agglutinin from winged bean (WBAI) in 0.02 M sodium phosphate and 0.15 M sodium chloride buffer have been investigated from 298.15 to 333.15 K by titration calorimetry and at the denaturation temperature by differential scanning calorimetry (DSC). WBAI is a dimer with two binding sites. The titration calorimetry yielded single-site binding constants ranging from 0.56 +/- 0.14 x 10(3) M-1 for Gal at 323.15 K to 7.2 +/- 0.5 x 10(3) M-1 for GalNAc at 298.15 K and binding enthalpies ranging from -28.0 +/- 2.0 kJ mol-1 for GalNAc at 298.15 K to -14.3 +/- 0.1 kJ mol-1 for methyl-beta-D-galactopyranoside at 322.65 K. The denaturation transition consisted of two overlapping peaks over the pH range 5.6-7.4. Fits of the differential scanning calorimetry data to a two-state transition model showed that the low temperature transition (341.6 +/- 0.4 K at pH 7.4) consisted of two domains unfolding as a single entity while the higher temperature transition (347.8 +/- 0.6 K at pH 7.4) is of the remaining WBAI dimer unfolding into two monomers. Both transitions shift to higher temperatures and higher calorimetric enthalpies with increase in added ligand concentration at pH 7.4. Analysis of the temperature increase as a function of added ligand concentration suggests that one ligand binds to the two domains unfolding at 341.6 +/- 0.6 K and one ligand binds to the domain unfolding at 347.8 +/- 0.6 K.

Carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus)

The carbohydrate binding specificity of the basic lectin from winged bean (Psophocarpus tetragonolobus) was investigated by quantitative precipitin analysis using blood group A, B, H, Le and I substances and by precipitation inhibition with various mono- and oligosaccharides. The lectin precipitated best with A1 substances and moderately with B and A2 substances, but not with H or Le substances. Inhibition assays of lectin-blood group A1 precipitation demonstration that A substance-derived oligosaccharides having the common structure: d-Ga1NAcα(1 → 3)d-Gal-(β1 → Image ) to a d-Glc, were the best inhibitors and about 8 and 4 times more active than d-Ga1NAc and d-Ga1NAcα(1 → 3)d-Ga1, respectively. A difucosyl A-specific oligosaccharide (A-penta), a monofucosyl (A-tetra) and a non-fucosyl containing (A5 II) oligosaccharide, d-Ga1NAcα(1 → 3)d-Ga1β(1 → 3)d-G1cNAc, had almost the same reactivity, suggesting that the fucose linked to the sub-terminal d-Ga1 or to the third sugar, d-GlcNAc, from the non-reducing end made no contribution to the carbohydrate binding. Although a terminal non-reducing d-Ga1NAc or d-Ga1 residue was indispensible for binding, the lectin bound not only to these terminal non-reducing galactopyranosyl residues, but also showed increased binding to oligosaccharides in which it was bonded to a sub-terminal d-Ga1 joined to a d-GlcNAc residue, as in blood group A or B substances. This defines the site, thus far, as complementary to a disaccharide plus the β linkage to the third sugar (d-Glc or d-GlcNAc) from the non-reducing end. The role of the β(1 → 3) or β(1 → 4) linkage of the sub-terminal non-reducing d-Gal to the d-GlcNAc requires further study.

Free energy of mixing of divalent basic oxides with silica

An expression derived for the free energy of mixing of a divalent basic oxide (MO) with SiO2 based on a model of silicate structure, takes into account the distribution of O2- (from MO) into the silica network, the mixing of silicate ions with O2- and the enthalpy of mixing. The resulting expression is ΔGmix=RT{N11n (2N1-N)2/4N1(1-N)+N21n N 2-N/1-N}, where N={(β+N1)-√(β+N 1)2-8βN1N2}/2β β=characteristic constant for the system N1=mol fraction of silica N2=mol fraction of MO. For the proper choice of β, calculated values of the activity of MO for the system PbO-SiO2, MnO-SiO2, FeO-SiO2 and CaO-SiO2 are in good agreement with experiment. The model predicts that the activity of the basic oxide decreases with increase in temperature.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Basic Income Grant in Namibia, a Viewpoint on the Proposal and on the Work of the BIG Coalition

This thesis studies the basic income grant proposal in Namibia. The proposal suggests a monthly grant of N$100 (approximately 10€) to all those Namibian citizens who do not receive the state pension. This thesis concentrates on the Basic Income Grant (BIG) Coalition and on its work. The formation and transformation of the coalition during the time period between 2003 and 2009 is analyzed with the help of data collected during two field work periods in 2008 and 2009. The data includes interviews, newspaper articles, observations and other background material. The analysis of this material is mainly conducted from organizational viewpoint. The final part of the thesis applies the results to the theory of Mosse, whose propositions about policy and practice will be discussed in relation to the basic income grant pilot project. The thesis argues that social legitimacy has been a vital resource for the work of the BIG Coalition and it has sought for it in various ways. The concept of social legitimacy originates from the resource dependence perspective of Pfeffer and Salancik, who propose that organizations are dependent on their environments, and on the resources provided by the surrounding environment. This thesis studies the concept of social legitimacy in the context of resource dependence theory. Social legitimacy is analyzed in the relations between the coalition and its environment, in the formation of the coalition, in the responses towards criticism, and finally in relation to the propositions concerning policy and practice. The work of the coalition in the pilot project will be analyzed through the propositions of Mosse concerning policy and practice. The results will describe and analyze key events in the formation of the BIG Coalition from the South African proposal until the end of the basic income pilot project. This BIG pilot project conducted in 2008-2009 is one of the most well-known activities of the coalition. The clashes between the coalition and its environment will be analyzed through four case studies. It will be shown that the project has been conducted in order to gain more legitimacy to the basic income grant proposal. The conclusion questions the legitimacy of the BIG Coalition as a research and development organization, and requests for more transparent research on the basic income proposal in Namibia.

Public and private technical assistance programs for non-industrial private forest landowners in the southern United States.

XVIII IUFRO World Congress, Ljubljana 1986.

Vain hätapua? : Taloudellinen avustaminen diakoniatyön professionaalisen itseymmärryksen ilmentäjänä

Financial Help Alone? Financial help as an exponent of professional diaconal work One essential form of helping people in the Evangelical Lutheran Church s diaconal work is providing economic aid. It can be seen as work which is in accordance with the spirit of the Church Order (4:3). One of the tasks of diaconal work, determined by the Church Order, is to help those whose distress is the greatest and who have no other source of help. This financial support has become a permanent and essential working method, which has also created tension of various kinds. Financial support has been criticized, especially when the support has been used to fill a gap in the social services provided by the government. It has been argued that diaconal work has been forced to take on responsibility for tasks that belong to the welfare state. The tensions involved in the financial support of diaconal work do not only concern the patching up and supplementing of the deficiencies in the welfare state s services but also the question of diaconal workers self-understanding of financial support and how it relates to their professionalism. In this thesis, I examine the experiences and visions diaconal workers have concerning financial support in their work with clients. The viewpoint of my work is the diaconal workers own experiences and interpretations of the meaning of financial support in customer service. In the articles of my thesis, I examined the meanings that diaconal workers gave to financial support in the aspects of work motivation, empowerment, expertise and tensions. The research material of my articles consists of three different data, which are theme interviews from diaconal workers, a survey from diaconal workers of Espoo and a diaconal barometer of 2009. I have analysed the theme interviews and the survey using qualitative content analysis. The results of my articles showed that diaconal workers motivation in tasks concerning economic aid was sustained by the nature and spiritual aspects of support activities. Work that supported empowerment through financial assistance meant influencing the client s personal life, community and local ties and structural circumstances of the surrounding society. Diaconal workers expertise in financial support work can be characterised as horizontal, which means that the expertise was built on acknowledging the client s dignity, the uniqueness of the client s life situation and listening to the client s own voice. Diaconal workers were also experts in community and area-based work. The tensions in financial support work are linked to its unofficial and undefined role in the field of social welfare and the inability of other aiding parties to respond to their duties. The results of my thesis on the experiences and visions of financial support reveal that it is multilateral and multidimensional. Diaconal workers used financial support to help the clients, taking into account their individual, communal, social and spiritual context. The professionalism of this financial support is reflectively related to the client s need of help and the spontaneity and unexpectedness of the situation. Support work was deeply bound to diaconal workers experiences of spirituality as the basic value in their work, the foundation of their idea of humanity and their method of helping others. In different tasks of financial support diaconal workers balanced between traditional, individual client work based on caritas and working methods which are based on supporting the individual s empowerment and active citizenship, as in postmodern social work. Diaconal workers experiences of financial support illustrated the transition or turning point in the professionalism of diaconal work, which involves finding one s own, stronger and clearer professional identity than earlier with respect to other helpers in society. Creating a unique identity is part of the empowerment process of diaconal work, in which it must define its professional role by itself. In postmodern pluralism and the fragmented context of diaconal activities, the question arose as to whether the spiritual traditions and traditional values of diaconal work support the modifications and adaptations needed in new, unpredictable situations. Diaconal work is said to be fast to react, able to predict changes and adapt to those changes. To preserve its sensitive reactive ability, also in the complex postmodern world, it must retain its own views and orientations. Otherwise, the distinctive values and traditions of diaconal work might sustain static diaconal work, employee-centeredness and a smug attitude when defining beneficiaries and needs, which highlights the paternalism of diaconal work. Such paternalism may complicate the progress of working methods which are based on empowerment and citizenship.

Thermodynamics of Phosphorus and Sulphur Removal during Basic Oxygen Steelmaking

Removal of impurity elements from hot metal is essential in basic oxygen steelmaking. Oxidation of phosphorus from hot metal has been studied by several authors since the early days of steelmaking. Influence of different parameters on the distribution of phosphorus, seen during the recent work of the authors, differs somewhat from that reported earlier. On the other hand, removal of sulphur during steelmaking has drawn much less attention. This may be due to the magnitude of desulphurisation in oxygen steelmaking being relatively low and desulphurisation during hot metal pre-treatment or in the ladle furnace offering better commercial viability Further, it is normally accepted that sulphur is removed to steelmaking slag in the form of sulphide only However, recent investigations have indicated that a significant amount of sulphur removed during basic oxygen steelmaking can exist in the form of sulphate in the slag under oxidising conditions. The distribution of sulphur during steelmaking becomes more important in the event of carry-over of sulphur-rich blast-furnace slag, which increases sulphur load in the BOF. The chemical nature of sulphur in this slag undergoes a gradual transition from sulphide to sulphate as the oxidative refining progresses.

Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.