Direct voltammetric observation of redox driven changes in axial coordination and intramolecular rearrangement of the phenylalanine-82-histidine variant of yeast iso-1-cytochrome c

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates. The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome. Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, DeltaS0'Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- --> Met80-Fe2+-His18. For the Phe82His variant mixed redox couple, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'Rxn = -52 J/mol.K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.     

A comparison of spectral and physicochemical properties of yeast iso-1 cytochrome c and Cys 102-modified derivatives of the protein

Derivatives of yeast iso-1 cytochrome c, chemically modified at Cys-102 (Cys-102 acetamide-derivatized monomer, Cys-102 thionitrobenzoate-derivatized monomer, Cys-102 S-methylated monomer, and the disulfide dimer), exhibit different spectral and physicochemical properties relative to the native, unmodified protein, depending on the nature of the modifying group. The results of proton NMR studies on the Cys-102 acetamide-derivatized monomer of iso-1 ferricytochrome c indicate that the conformational characteristics of the heme environment in this protein derivative are intermediate between those of the unmodified monomer and disulfide dimer forms of the protein. Measurements of the pKa of the alkaline transitions of the five forms of iso-1 ferricytochrome c provided values of 8.89, 8.82, 8.67, 8.47, and 8.50 for the unmodified monomer, S-methylated monomer, acetamide-derivatized monomer, thionitrobenzoate-derivatized monomer, and disulfide dimer, respectively. The results of proton NMR studies of the reduced form of these proteins suggest that the heme environments of the unmodified monomer and disulfide dimer derivatives of iso-1 ferrocytochrome c are similar and indicate that treatment of the thionitrobenzoate-derivatized and disulfide dimer forms of the protein with sodium dithionite results in cleavage of the disulfide bonds at position 102. Circular dichroism studies reveal that only the disulfide dimer form of iso-1 ferricytochrome c exhibits a Soret CD spectrum which differs from the native, unmodified monomer in that the intensity of the negative band at approximately 420 nm is diminished in the spectrum of the dimer relative to the spectrum of the monomer. Soret CD spectra of the ascorbate-reduced form of all protein derivatives are similar. The process of "autoreduction" of yeast iso-1 ferricytochrome c is shown to occur in the absence of a free sulfhydryl group at position 102 and is exacerbated under moderately high pH conditions. These results are suggestive of the presence of a redox-active amino acid, perhaps a tyrosine, in yeast iso-1 cytochrome c.     

The Win1 mitotic regulator is a component of the fission yeast stress-activated Sty1 MAPK pathway

Women's anger experience has been poorly understood and insufficiently researched. Yet the emotion of anger is vitally important to women's physical and mental health, and to the quality of their relationships. This phenomenological study was undertaken as an expansion and extension of the Women's Anger Study, the first large survey of the genesis, manifestations and correlates of anger in American women. Although the earlier study contributed to understanding of anger, a deeper examination of the context and meanings of anger experience was sought. Twenty-nine Caucasian women ranging in age from 21 to 66 years were interviewed. Illustrative occupations ranged from homemaker, student, waitress to business executive, professor, and human service professional. Analysis involved thematizing by the researchers independently and within a multidisciplinary phenomenological research group. The thematic structure of women's anger involved a building over time of a confusing mixture of feelings (hurt, frustration, disillusionment) precipitated by a violation of the core values of the self. The precipitant of anger was unfair and/or disrespectful treatment or lack of reciprocity in relationships. When the anger was confined within self, the woman felt helpless and powerless. However, powerlessness was also evident when anger was externalized in an outburst. To the study participants, an angry outburst meant a loss of control rather than ability to achieve control. Women reported a sense of power when using anger to restore justice, respect, and relationship reciprocity. Clinicians can assist women to reflect on their core values and use the power of their anger effectively. Further studies are in progress to examine the relevance of these findings for women of other races and cultural contexts.     

Effect of squalestatin 1 on the biosynthesis of the mevalonate pathway lipids

The effects of squalestatin 1 on rat brain and liver homogenates and on Chinese hamster ovary tissue culture cells have been investigated. This compound effectively inhibits squalene biosynthesis in a highly selective manner. Cytoplasmic farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases are not affected, which is also the case for microsomal cis-prenyltransferase. In tissue culture cells, squalestatin 1 inhibits cholesterol biosynthesis completely, but does not alter dolichol synthesis or protein isoprenylation to a great extent. Incorporation of [3H]mevalonate into ubiquinone-9 and -10 increases 3-4-fold, probably as a result of increased synthesis of this lipid. Squalestatin 1 appears not only to be an effective inhibitor of cholesterol biosynthesis, but also to be more specific than other inhibitors used earlier in various in vitro and in vivo systems.     

UDP-GlcNAc:Galbeta1-->3GalNAc (GlcNAc to GalNAc) beta1-->6N-acetylglucosaminyltransferase holds a key role on the control of CD15s expression in human pre-B lymphoid cell lines

Distraction osteogenesis has become an important technique to treat craniofacial skeletal dysplasia. In this study, the technique of maxillary distraction with a rigid external distraction device is presented. Cephalometric results in the first 14 consecutive patients are analyzed. The study sample consisted of 14 patients with various cleft types and maxillary hypoplasia treated with the rigid external distraction technique. Analysis of the predistraction and postdistraction cephalometric radiographs revealed significant skeletal maxillary advancement. All patients had correction of the maxillary hypoplasia with positive skeletal convexity and dental overjet after maxillary distraction. The morbidity for the procedure was minimal. Surgical and orthodontic procedures are thoroughly described.     

Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1-->6GalNAcalpha core structure as a tumor-associated antigen

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.     

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.     

Effect of cross-flow on the frequency of bubble formation from a single-hole nozzle

Bubble formation from a single-hole nozzle placed vertically upward in a rotating water bath was investigated using a high-speed video camera. Air was used as the working gas. The measured values of the frequency of bubble formation, f _B, were compared with those observed in a stationary bath, f _B0. The velocity of cross-flow, ν_θ, affected the bubble formation significantly when it exceeded a critical value, ν_θc . The ratio of f _B to f _B0 was unity for ν_θ≦ν_θc , but it changed in a complex manner for ν_θ>ν_θc . In the latter case, when the air flow rate Q _g was relatively low, f _B/f _B0 became larger than unity irrespective of Q _g, and an empirical correlation of f _B/f _B0 was proposed as a function of ν_θ and the inner diameter of the nozzle, d _ni. As the gas flow rate increased, f _B/f _B0 decreased monotonically and became smaller than unity, and an empirical correlation of f _B/f _B0 was derived as a function of Q _g, ν_θ, and d _ni. These empirical correlations could approximate the measured values of f _B/f _B0 within a scatter of −15 to +20 pct.     

Effect of the physiological properties of yeast strains on their formation of higher alcohols and aldehydes and on the composition of nitrogen compounds in the fermented wort

Dynamics of formation of by-products was studied with the following yeast races: Sacch. cerevisiae, strain Odesskaya-14 (baker's yeast); Sacch. vini, strain Prikumskaya 80/9 (wine yeast). The yeast cultures were found to be very similar by the rate of biomass accumulation, ethanol production, and the fractional composition of nitrogen compounds. The concentration of accumulated higher alcohols depended on the mass of yeast cells, their growth rate, and the duration of cultivation.     

Effect of argon injection on the rim formation in a section continuous casting

The effect of argon blowing into a steel stream on the stability of continuous casting is studied. Physical modeling and mathematical simulation are performed to find the parameters of argon blowing into a steel stream during the casting of a section billet.     

Effect of argon injection on the rim formation in a section continuous casting

The effect of argon blowing into a steel stream on the stability of continuous casting is studied. Physical modeling and mathematical simulation are performed to find the parameters of argon blowing into a steel stream during the casting of a section billet. Original Russian Text ? E.V. Shtepan, G.A. Red’ko, V.E. Ukhin, A.V. Naumenko, E.Yu. Zhiboedov, 2007, published in Elektrometallurgiya, 2007, No. 6, pp. 20–23.     

Effect of acidic pH on the structure and lipid binding properties of porcine surfactant protein A. Potential role of acidification along its exocytic pathway

Pulmonary surfactant protein A (SP-A) is synthesized by type II cells and stored intracellularly in secretory granules (lamellar bodies) together with surfactant lipids and hydrophobic surfactant proteins B and C (SP-B and SP-C). We asked whether the progressive decrease in pH along the exocytic pathway could influence the secondary structure and lipid binding and aggregation properties of porcine SP-A. Conformational analysis from CD spectra of SP-A at various pH values indicated that the percentage of alpha-helix progressively decreased and that of beta-sheet increased as the pH was reduced. The protein underwent a marked self-aggregation at mildly acidic pH in the presence of Ca2+, conditions thought to resemble those existing in the trans-Golgi network. Protein aggregation was greater as the pH was reduced. We also found that both neutral and acidic vesicles either with or without SP-B or SP-C bound to SP-A at acidic pH as demonstrated by co-migration during centrifugation. However, the binding of acidic but not neutral vesicles to SP-A led to 1) a striking change in the CD spectra of the protein, which was interpreted as a decrease of the level of SP-A self-aggregation, and 2) a protection of the protein from endoproteinase Glu-C degradation at pH 4.5. SP-A massively aggregated acidic vesicles but poorly aggregated neutral vesicles at acidic pH. Aggregation of dipalmitoylphosphatidylcholine (DPPC) vesicles either with or without SP-B and/or SP-C strongly depended on pH, being progressively decreased as the pH was reduced and markedly increased when pH was shifted back to 7.0. At the pH of lamellar bodies, SP-A-induced aggregation of DPPC vesicles containing SP-B or a mixture of SP-B and SP-C was very low, although SP-A bound to these vesicles. These results indicate that 1) DPPC binding and DPPC aggregation are different phenomena that probably have different SP-A structural requirements and 2) aggregation of membranes induced by SP-A at acidic pH is critically dependent on the presence of acidic phospholipids, which affect protein structure, probably preventing the formation of large aggregates of protein.     

Effect of electrodynamic phenomena in arc furnaces on the meniscus formation in a liquid metal

The main laws of the electrodynamic action of an electric arc on the surface of the liquid metal bath in electric arc furnaces of various types are considered.     

Effect of the design of a supply nozzle on the ingot formation during semicontinuous casting of copper

The semicontinuous casting of copper using a T-joint submerged nozzle with various configurations of its internal contour and a various number of outlet holes is considered. The results of physical modeling and commercial tests are used to choose the design of the submerged nozzle that meets the conditions of copper casting under industrial conditions as much as possible.     

Effect of an electric potential on the formation of a dislocation structure during creep of aluminum

The effect of an electric potential on the formation of a dislocation substructure at the stage of steady-state creep in commercial-purity aluminum is analyzed using the theoretical concepts of steady-state creep and the equations describing a dislocation ensemble with allowance for an applied electric potential. The effect of an electric potential on the creep rate and acceleration is considered through the mechanism of the effect of the potential on the surface energy of aluminum.