Large-scale Structural Changes Research Articles

Physical Factors Affecting the Storage Stability of Freeze-Dried Interleukin-1 Receptor Antagonist: Glass Transition and Protein Conformation

The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56°C, during several weeks of storage at temperatures above and below the samples’ Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 ± 2.5°C. With infrared spectroscopy, we determined that the protein lyophilized with ≤1% sucrose was unfolded in the initial dried solid. In contrast, in those formulations with ≥5% sucrose, conformational change was inhibited during lyophilization. When stored at 50°C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation.

HRM in the New Economic Environment: An Empirical Study of India

This paper is developed from the second author's ongoing Ph.D. research, which focuses on the managerial thinking (personnel specialists) about strategic management of human resources from a cross‐cultural viewpoint between India and Britain. The Indian Personnel Specialists are under a severe pressure to bring about large scale structural changes in their organisations to cope with the challenges thrown by the recent liberalised economic policies. The role of Human Resource (HR) function has become more important than ever in such conditions. An attempt is therefore made to analyse the HR function in India in the changing economic environment. The influence of a number of national and contingent variables on the HR function is therefore studied to place it against the worldwide patterns of Human Resource Management (HRM) practices.

Electron Transport Phenomena in NiS2−xSex Single Crystals

AbstractStructural studies, electrical resistivity, and Seebeck coefficient measurements are reported in the range 4.2 − 300 K for single crystals of NiS2−xSex (0 ≤ x ≤ 0.71) grown from a Te melt. Over the entire temperature and composition ranges there are no large scale structural changes concomitant to a variety of magnetic ordering phenomena, and to a changeover from insulating to metallic characteristics as x increases. Thus, the evolution in transport characteristics with x can be studied without interference from the lattice; moreover, the electron count is unaffected by substitution of Se for S. The existence of anomalous peaks in resistivity as a function of temperature is attributed to significant electron correlation phenomena which allow the entropy of charge carrier to play a dominant role. The complex temperature dependence of the Seebeck coefficient is attributed to the participation of both electrons and holes in charge transport.

NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48.

The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2).

Longitudinal distribution of Trichoptera in the Loire River (France): simple ordination methods and community structure

The longitudinal distribution of Trichoptera at 37 sampling sites along 800 km of the Loire River (France) was analyzed in order to better understand large-scale changes in community structure occurring on a stream continuum. Different simple ordination methods — centered PCA and Correspondence Analysis — were performed to investigate these changes. Both analyses showed that a major change occurs in the community in the upper reaches of the Loire River between the Serre de la Fare and Vorey sampling sites with the appearance of Hydropsyche contubernalis and H. Exocellata. Even though other changes in community structure could exist, revealed either by Correspondence Analysis at the first sampling sites (crenal), or by PCA at sampling sites influenced by dams, this Trichoptera community appears to be stable enough in the latter part of the Loire River with the addition of a few species, Hydropsyche bulgaromanorum, Ecnomus tenellus and E. deceptor, rather than changes in species. The results are discussed in the light of different concepts in stream ecology, and the validity of such data analyses to describe some community patterns.

Resolution of three structural states of spin-labeled myosin in contracting muscle

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.

Features of the solar wind large-scale structure in the different periods of solar activity based on the variations of cosmic rays

The relations between the energy spectrum exponent γ of cosmic ray isotropic intensity variations ( δD(R) D (R) ∝ R −γ ) and the parameter α which characterizes the diffusion coefficient's χ dependence on the cosmic ray particle's rigidity R ( χ∝ R α ) has shown ( α ∝ γ). The large-scale structural changes of the magnetic inhomogeneities of solar wind in the different periods of solar activity is the general mechanism of 11-year variations of cosmic rays above energy 1–2 GeV. The energy spectrum exponent γ of cosmic ray isotropic intensity variations can be considered as a important tool for the investigation of the temporal and spatial changes of the large-scale fluctuations in the interplanetary magnetic field strength.

Changes in the relative occupancy of metal-binding sites in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ATPase enzyme in the presence of terbium: a time-resolved, resonance x-ray diffraction study

Time-resolved, terbium resonance x-ray diffraction experiments have provided the locations of three different high-affinity metal-binding/transport sites on the Ca2+ATPase enzyme in the profile structure of the sarcoplasmic reticulum (SR) membrane. By considering these results in conjunction with the known, moderate-resolution profile structure of the SR membrane (derived from nonresonance x-ray and neutron diffraction studies), it was determined that the three metal-binding sites are located at the "headpiece/stalk" junction in the Ca2+ATPase profile structure, in the "transbilayer" portion of the enzyme profile near the center of the membrane phospholipid bilayer, and at the intravesicular surface of the membrane profile. All three metal-binding sites so identified are simultaneously occupied in the unphosphorylated enzyme conformation. Phosphorylation of the ATPase causes a redistribution of metal density among the sites, resulting in a net movement of metal density toward the intravesicular side of the membrane, i.e., in the direction of calcium active transport. We propose that this redistribution of metal density is caused by changes in the relative binding affinities of the three sites, mediated by local structural changes at the sites resulting from the large-scale (i.e., long-range) changes in the profile structure of the Ca2+ATPase induced by phosphorylation, as reported in an accompanying paper. The implications of these results for the mechanism of calcium active transport by the SR Ca2+ATPase are discussed briefly.

The Ti Plasmid from the Wide Host Range Agrobacterium vitis Strain Tm4: Map and Homology with Other Ti Plasmids

The octopine/cucumopine (o/c) strains of Agrobacterium vitis are divided into limited (LHR) and wide host range (WHR) strains. We present here the first map of a Ti plasmid from a WHR o/c strain, Tm4. pTiTm4 (252 kb) has homology with the octopine Ti plasmid pTiAch5, the nopaline plasmid pTiC58, and the Ri plasmid pRiHRI. Approximately 155 kb of pTiTm4 is homologous with the LHR o/c Ti plasmids pTiAB3 and pTiAg57, the remaining part is pTiTm4-specific. Our data suggest that the evolution of the o/c Ti plasmids was accompanied by large-scale changes in plasmid structure. The origin of replication of pTiTm4 was localized by a functional assay.

Molecular analysis of rat embryo cell transformants induced by alpha-particles.

An immortal cell line was established by transfecting a myc oncogene into rat embryo cells (REC:myc). This cell line was diploid, contact inhibited and grew well in culture. Exposure to a single 200 cGy dose of 6 MeV alpha-particles transformed these cells with a frequency of focus formation of approximately 3.6 x 10(-4) compared with a transformation frequency of < 7.8 x 10(-6) for primary cultures of REC. Isolates of alpha-particle-induced REC:myc (REC:myc:alpha) foci displayed anchorage-independent growth in soft agar and were tumourigenic in nude mice. Molecular studies demonstrated no alteration of gene structure or expression of the transfected or of the endogenous c-myc genes. Similarly, there was no alteration of the structure of Ha-ras, Ki-ras, or N-ras. The expression of Ha-ras, Ki-ras, N-ras and raf was not altered significantly. Assay for dominant oncogenes via DNA-mediated gene transfer into NIH3T3 cells was positive for nine of 13 REC:myc:alpha transformants. All NIH3T3 isolates contained bands hybridizing to rat repetitive DNA. NIH3T3 transformants from a tertiary round of transfection were analysed by Southern blot analysis for the presence of Ki-ras, N-ras, raf, trk, abl, fms, src, mos, fos, sis, fps, erbA, erbB or neu oncogenes of REC origin, and none were detected. Tertiary NIH3T3 transformants from three REC:myc:alpha transformants contained bands corresponding to Ha-ras but no point mutations were identified at the known hotspots of exons 1 or 2 of the donor REC:myc:alpha transformants. The inactivation of the tumour suppressor genes Rb, and p53, and the anti-metastasis gene, nm23, was evaluated by Southern and Northern hybridization analysis. Southern blots demonstrated that at least one allele of Rb, p53 and nm23 was present and no large scale structural changes were detected. No expression of Rb or p53 was detected in REC:myc or the alpha-particle-induced REC:myc transformants. The expression of nm23 was not altered in the transformed cell lines. While the analysis of the role of tumour suppressor gene inactivation in radiation-induced cell transformation is only in the initial stages, the results of DNA-mediated gene transfer into NIH3T3 cells suggest that unidentified dominant oncogenes are associated with alpha-particle-induced transformation in vitro.

Secular evolutionary trends in ellipticals and bulges due to mergers

Using basically the impulsive approximation and a modification of the method used by Alladin S.M., (1965, Ap.J.,141, 768), and described in detail in Chatterjee T.K., (1990, IAU Col.,124, 519, 569) we study the evolution of binary interacting galaxies, leading ultimately to mergers. Each collision is characterised by the initial separation between the galaxies and the relative velocity therein. In each case the orbital evolution and largescale structural changes in the galaxies are studied by taking into account the change in relative velocity due to dynamical friction, leading ultimately to mergers. The evolution is considered from a time when the gravitational interaction between the progenitor pairs is physically significant (Chatterjee, 1992, Astroph. Sp.Sc., in press).

Size and mobility of sodium dodecyl sulfate—bovine serum albumin complex as studied by dynamic light scattering and electrophoretic light scattering

Electrophoretic light scattering and dynamic light scattering methods were applied to measure the electrophoretic mobilities and radii of the complexes of bovine serum albumin (BSA) with sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB). In the phosphate buffer of pH 7.0 and ionic strength 0.014, the mobility of BSA, μ BSA, was −1.7 × 10 −4 cm 2 s −1 V −1. The negative magnitude of μ BSA sharply increased at low SDS concentrations below 2 m M. The negative mobility sharply increased again above 5 m M SDS and reached −4.7 × 10 −4 cm 2 s −1 V −1 at 8 m M. In the DTAB solution, μ BSA remained negative below 6 m M. It crossed zero mobility at 6 m M DTAB and became positive beyond this concentration. The mobilities of BSA—SDS and BSA—DTAB complexes attained at high surfactant concentrations were appreciably smaller than those of the corresponding surfactant micelles. On the other hand, the effective hydrodynamic radius of BSA, R BSA, was estimated to be 3.1 nm. The magnitude of R BSA increased up to 6.0 and 5.2 nm with increases of SDS and DTAB concentrations, respectively. The changes in these μ BSA and R BSA values occurred in the surfactant concentration ranges where the secondary structure of BSA was disrupted. The secondary structural change of the protein appeared likely to accompany a large-scale tertiary structural change.

Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Structural and functional changes.

As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

Crime and the transformation of capitalism

This study investigates factors that enhance criminal behavior. Typically, explanations for increasing crime rates discuss various familial and community attributes; i.e., single parent households and poverty rates. When crime has increased, these attributes are cited as being responsible. This we believe is an incorrect assumption. In particular, we argue the factors that are cited by these authors merely reflect transformations within the greater economic structure. As a result, the familial and community attributes suggested as causing crime are seen as intervening variables; which are either attentuated or amplified in their ability to control criminal behavior by the condition of the economy itself. Within this study, five urban cities (New York, Detroit, Newark, Boston, and Philadelphia) are observed over a twenty eight year period. Structural change is measured by industrial employment and crime is measured by three indices: burglary, robbery and murder. Our investigation reveals a strong relationship between declining industrial employment and increasing crime rates. In addition, we find that the magnitude of this relationship is subject to variation. This variation is dependent upon whether or not the industrial decline experienced was steady or vacilating between high and low levels. These findings support the contention that familial units and other communal organizations are merely intervening variables between large-scale structural changes and criminal behavior. Moreover, it suggests that unless anti-crime efforts (increasing the size of the police forces, increasing state expenditures for law enforcement, etc.) are accompanied by strategies of industrial reorganization, they will have no impact on reducing crime rates.

Fibers spun from blends of different molecular weights of polypropylene

AbstractFibers spun from blends of small percentage of plastic grade polypropylene (HMPP) with fiber grade polypropylene (PP) are studied for drawing behavior. A factorial design of experiment is used for a two‐stage drawing process with variables, such as percent of HMPP component, first stage draw ratio and temperature, and second stage temperature. Optimization is carried out for breaking stress and modulus of drawn filaments. Breaking stress of up to 0.74 GPa and initial modulus of 7.34 GPa is possible by such an optimization process. These properties are observed for 6% HMPP blend composition. Heat setting of drawn filaments show little changes up to 140°C heat‐setting temperature. Large scale structural changes with rapid drop in mechanical properties is observed for 150‐160°C heat‐set samples.

Social movement research in the United States: A European perspective

In the effort to create a general, encompassing theory of social movements, a flurry of proposals for “synthesis” have recently been submitted. At first, suggestions were made to reintroduce social-psychological perspectives to the resource mobilization approach (Klandermans, 1984; Ferree and Miller, 1985). Now, the integration of even the resource mobilization and classical perspectives is urged (McAdam, McCarthy and Zald, 1988; Rule, 1989). And in the international debate, a synthesis of European (structural) and American (resource mobilization) traditions is proposed. (Kriesi, 1988; p. 364; Klandermans and Tarrow, 1988; Tarrow, 1991). These propositions, no doubt, reflect researchers’ growing awareness of the particular limitations within each perspective to fully account for the emergence, dynamic, and characteristics of these movements. Thus, it is hoped that the systematic silences within the theory of resource mobilization (RM) on the role of norms, beliefs, or emotions might be filled by a return to some of the explanations offered within the collective behavior or mass society tradition. Or, in a somewhat parallel fashion, that the recent European new social movement theories, which link social movements to large-scale structural or cultural change, might nicely complement the American resource mobilization approach, as the latter focuses more exclusively on mobilization processes at the group and individual level.

Non-additivity of sequence-specific enzyme-DNA interactions in theEcoRI DNA methyltransferase

We describe a novel strategy to characterize protein-DNA interactions involving monomeric enzymes such as DNA methyltransferases (Mtases). This strategy is applied to our investigation of the EcoRI DNA Mtase, which binds its double stranded recognition site 5'-G-AATTC-3' and methylates the central adenosine of each strand using S-adenosyl-L-methionine as the methyl donor. We show that prior methylation of adenosine in either strand does not perturb catalysis. In contrast, substrates substituted with deoxyinosine at either guanosine position (T-BMI5 and TI5-BM) show the minor groove residing N2 amino group of both guanosines contribute to DNA recognition since specificity constants for the modified substrates are reduced 13 and 39 fold. Similar analysis of a substrate containing deoxyinosine at both positions (TI5-BMI5) clearly shows that some communication occurs between the sites. To determine the extent to which structural changes in the DNA alone contribute to this lack of additivity, we performed DNA melting analysis of the singly and doubly substituted substrates, and also found non-additivity. Although our functional and structural analyses suggest that deoxyinosine incorporation causes long range conformational effects, the similarity of KmAdoMet for all substrates suggests that no large-scale structural changes occur in the Mtase-DNA-AdoMet complex. Our results support the following conclusions: 1) The non-additivity shown in this system contrasts with the widespread demonstration of additivity involving repressors [Lehming et al., 1990; Takeda et al., 1989; Ebright et al., 1987], suggesting that sequence discrimination by enzymes may involve more complex mechanisms. Further, this non-additivity precludes quantitative assignment of individual interactions and we suggest that future analyses of this and related enzyme systems with base analogs include detailed information about the long range structural consequences of individual substitutions. 2) Although TI5-BM and T-BMI5 are shown to be radically different by thermodynamic analysis, the similar specificity constants with the Mtase suggest that the underlying structural differences (e.g., altered helical parameters of the DNA) are not critical for sequence-recognition. 3) The significance of minor groove Mtase-DNA interactions to specificity is confirmed.

Recent neutron scattering studies of muscle contraction and its control

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied. For free chymotrypsin-generated S1 it was found that Rg = 4.0 +/- 0.15 nm, while for papain-generated S1 it was found that Rg = 4.6 +/- 0.2 nm. Upon binding of papain-generated S1 to actin at low NS1/N actin ratios, the change in Rg in difference experiments was delta Rg = 0.05 +/- 0.15 nm. This lack of significant change in Rg in the very low-s domain confirms and extends our earlier neutron scattering work in the higher-s domain. The longest chords of S1, as well as shorter ones, are not significantly altered upon actin binding. These results indicate that muscle contraction does not occur as a result of large-scale changes in S1 structure. In actin-Tm complexes, a measurement of the mean cross-helix separation, d, of Tm molecules has been made using neutron scattering. With deuterated actin matched out in 93% D2O buffer, it was found that d = 7.9 +/- 0.3 nm. This value is in good agreement with a model based on Tm crystallography and also with recent electron microscopy results. These experiments demonstrate the feasibility and value of neutron diffraction and scattering techniques in the study of muscle contraction and its control. One can expect that the further employment of emerging cell biology techniques for generating deuterated proteins will aid our understanding of muscle in the future.

Large-scale structural changes in the sarcoplasmic reticulum ATPase appear essential for calcium transport

Model refinement calculations utilizing the results from time-resolved x-ray diffraction studies indicate that specific, large-scale changes (i.e., structural changes over a large length scale or long range) occur throughout the cylindrically averaged profile structure of the sarcoplasmic reticulum ATPase upon its phosphorylation during calcium active transport. Several physical-chemical factors, all of which slow the kinetics of phosphoenzyme formation, induce specific, large-scale changes throughout the profile structure of the unphosphorylated enzyme that in general are opposite to those observed upon phosphorylation. These results suggest that such large-scale structural changes in the ATPase occurring upon its phosphorylation are required for its calcium transport function.

Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase.

Diffuse X-ray-scattering data give evidence for large-scale structural change in pig muscle 3-phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3-phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1-nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3-phosphoglycerate kinase. When, instead of methylation, the two reactive thiol groups of pig muscle 3-phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its 'apo' and 'holo' forms do not differ within limits of experimental error. Thus, a correlation exists between the activity of 3-phosphoglycerate kinase and its substrate-induced large-scale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3-phosphoglycerate kinase.