Spectroscopically Distinct Cobalt(II) Sites in Heterodimetallic Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e: Characterization of Substrate Binding

The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT. These parameters correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited 59Co hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322−5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with geff values of 2.08, 3.15, and 6.15 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP [Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc. 119, 1923−1933; Chen, G., et al. (1997) Biochemistry 36, 4278−4286]

Lipopolysaccharide and toll-like receptor 4 in dogs with congenital portosystemic shunts

Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased portal vein perfusion, liver growth and clinical improvement. Portal lipopolysaccharide (LPS) is implicated in liver regeneration via toll-like receptor (TLR) 4 mediated cytokine activation. The aim of this study was to investigate factors associated with LPS in dogs with CPSS. Plasma LPS concentrations were measured in the peripheral and portal blood using a limulus amoebocyte lysate (LAL) assay. LPS concentration was significantly greater in the portal blood compared to peripheral blood in dogs with CPSS (P = 0.046) and control dogs (P = 0.002). LPS concentrations in the peripheral (P = 0.012) and portal (P = 0.005) blood of dogs with CPSS were significantly greater than those of control dogs. The relative mRNA expression of cytokines and TLRs was measured in liver biopsies from dogs with CPSS using quantitative PCR. TLR4 expression significantly increased following partial CPSS attenuation (P = 0.020). TLR4 expression was significantly greater in dogs that tolerated complete CPSS attenuation (P = 0.011) and those with good portal blood flow on pre-attenuation (P = 0.004) and post-attenuation (P = 0.015) portovenography. Serum interleukin (IL)-6 concentration was measured using a canine specific ELISA and significantly increased 24 h following CPSS attenuation (P < 0.001). Portal LPS was increased in dogs with CPSS, consistent with decreased hepatic clearance. TLR4 mRNA expression was significantly associated with portal blood flow and increased following surgery. These findings support the concept that portal LPS delivery is important in the hepatic response to surgical attenuation. Serum IL-6 significantly increased following surgery, consistent with LPS stimulation via TLR4, although this increase might be non-specific

EPR Studies on the Mono- and Dicobalt(II)-Substituted Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e. Insight into the Catalytic Mechanism of Dinuclear Hydrolases

The structure and function of the prototypical dinuclear hydrolase, namely, the aminopeptidase from Aeromonas proteolytica (AAP), was probed by EPR spectroscopy of the mono- and dicobalt(II)-substituted derivatives. A new systematic protocol for the interpretation of Co(II) EPR spectra is described and the S = 3/2 spin states of the Co(II)-substituted forms of the enzyme have been characterized. This protocol allows the simulation of line shape using theoretically allowed geff values corresponding to an isotropic greal value. In addition, the gross distortion of EPR spectra of high-spin S = 3/2 Co(II) ions has been investigated, and the effects of saturation on the line shapes and on simulation-derived spectral parameters are discussed. For [Co-(AAP)], a distinctive EPR signal was observed in which the hyperfine pattern due to 59Co was not centered on the low-field absorption feature, and the signal could not be simulated as a single species. Subtraction of EPR spectra recorded at different temperatures revealed that two species were, in fact, present in samples of [Co-(AAP)]. The first species was a relatively featureless axial signal with geff values of 5.75, 4.50, and 2.50. These values correspond to an Ms = |±1/2〉 ground-state transition with greal = 2.57 and E/D = 0.08. The second species had geff values of 6.83, 2.95, and 1.96 and exhibited a characteristic eight-line 59Co hyperfine pattern with Az = 7.2 mT. The observed 59Co hyperfine lines were simulated in both line width as well as signal intensity for the first time. These parameters correspond to the Ms = |±1/2〉 ground-state transition with greal = 2.57; however, the signal exhibited marked rhombicity (E/D = 0.28), consistent with a highly distorted tetrahedral Co(II) species. The possibility that the spectrum could be due to contributions from the Ms = |±1/2〉 and Ms = |±3/2〉 doublets of a single spin system was investigated, but subtraction of spectra recorded at various temperatures clearly indicated that the features at g = 2.95 and g = 1.96 were correlated with the feature at g = 6.83. In addition, at temperatures above 15 K, the signal intensity rapidly decreases and the signal is lost. The EPR spectrum of [CoCo(AAP)] reveals a relatively featureless signal that was simulated as a single species with geff(1,2,3) values of 5.10, 3.85, and 2.19; Ms = |±1/2〉; greal = 2.25; E/D = 0.095. The intensity of the observed signal for [CoCo(AAP)] corresponded to 0.13 spin/mol of Co(II). These data strongly suggest that the two Co(II) ions in the active site of AAP experience significant spin−spin interaction and are either antiferromagnetically or ferromagnetically coupled. Perpendicular mode EPR titration of apo-AAP with Co(II) revealed a low-field signal extending out of zero-field in samples with more than 1 equiv of Co(II) added. This type of EPR absorption is indicative of an integral spin system. Coincident with the appearance of the low-field perpendicular mode signal was the appearance of a parallel mode EPR signal with g ∼ 12. These data represent the first definitive evidence for ferromagnetic coupling between two high-spin S = 3/2 Co(II) ions in a dinuclear center. The effect of pH, added peroxide, and the coordination of the competitive inhibitor 1-butaneboronic acid (BuBA) on the signal both confirm the origin of the signal and provide important mechanistic information for this novel dicobalt(II) active site cluster. Based on the present study and the available literature data, a detailed mechanism of action is proposed for AAP

The Use of Scaffolding Technique to Teach Recount Text Writing

The purpose of this research was to investigate the effectiveness of using scaffolding technique in teaching writing especially recount text writing to Year-10 Students of SMA Karya Sekadau in Academic Year 2015/2016. It was a pre-experimental study with a single group pre-test post-test design. The sample was Year-10 students that consisted of 29 students. The data were collected by giving pre-test before the treatment and post-test after the treatment in writing test. The test was performance test in written form.. Pre-test and post-test were the same test with the same command. The data were analyzed by Effect Size (ES) formula. The finding showed that the effect size of teaching recount text writing by using scaffolding technique was 1.210 (ES > 0.5) and categorized high

Reading materials for junior high school, contributions to American life made by members of minority groups

Thesis (Ed.M.)--Boston University, 1948. This item was digitized by the Internet Archive

The pituitary TGFb1 system as a novel target for the treatment of resistant prolactinomas

Prolactinomas are the most frequently observed pituitary adenomas and most of themrespond well to conventional treatment with dopamine agonists (DAs). However, a subsetof prolactinomas fails to respond to such therapies and is considered as DA-resistantprolactinomas (DARPs). New therapeutic approaches are necessary for these tumors.Transforming growth factor b1 (TGFb1) is a known inhibitor of lactotroph cell proliferationand prolactin secretion, and it partly mediates dopamine inhibitory action. TGFb1 is secretedto the extracellular matrix as an inactive latent complex, and its bioavailability is tightlyregulated by different components of the TGFb1 system including latent binding proteins,local activators (thrombospondin-1, matrix metalloproteases, integrins, among others), andTGFb receptors. Pituitary TGFb1 activity and the expression of different components of theTGFb1 system are regulated by dopamine and estradiol. Prolactinomas (animal models andhumans) present reduced TGFb1 activity as well as reduced expression of several componentsof the TGFb1 system. Therefore, restoration of TGFb1 inhibitory activity represents a noveltherapeutic approach to bypass dopamine action in DARPs. The aim of this review is tosummarize the large literature supporting TGFb1 important role as a local modulator ofpituitary lactotroph function and to provide recent evidence of the restoration of TGFb1activity as an effective treatment in experimental prolactinomasFil: Recouvreux, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Cedars Sinai Medical Center; Estados UnidosFil: Camilletti, María Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rifkin, Daniel B.. University of New York; Estados UnidosFil: Diaz, Graciela Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Литературные портреты творческих личностей в возрасте в сборнике пьес Тины Хау «Прибрежные волнения»

Considering increased average life expectancy, the  world-wide demographical crisis and discrimination of the elderly, the article studies strategies of aging and various forms of adaptation to changes in late adulthood in fiction. The goal of the study is examination of representations of the fictional elderly artists in a collection of  plays by  Tina Howe (1989). The  author of  the article analyzes the  interaction of  art and late adulthood in  Museum, Painting Churches and Coastal Disturbances. Contemplation of  art and involvement into artistic activities help establish positive images of aging in three dramas. Life review fragments, discourse of death drive, discussion of  elderly suicide and self-stereotyping are defined as  gerontological markers of  aging. The  plays in question build an ambivalent solidarity-conflict model between aging parents and adult child. The involvement into artistic activities becomes a way to cope with aging in lives of the elderly characters.Актуальність даної розвідки зумовлена збільшенням середньої тривалості життя, світовою демогра- фічною кризою та дискримінацією людей літнього віку, що знаходить відображення у сучасній белетрис- тиці, зокрема у драматургії. Метою статті є вивчення репрезентацій митців літнього віку в збірці ко- медій сучасної американської жінки-драматурга Тіни Хау 1989 р. Динаміка геронтогенезу представлена моделюванням позитивних образів, залучених до споглядання або участі у творах мистецтва, у трьох п’єсах. Виявлені наступні геронтологічні маркери: фрагменти перегляду життя у формі спогадів, тана- тологічний дисурс, розмірковування про суїцид серед представників пізньої дорослості та репліки само- стереотипізації. Встановлено амбівалентну модель солідарності-конфлікту у розробці мережі стосун- ків між старіючими персонажами та їхніми дорослими дітьми.Актуальность данной статьи обусловлена увеличением средней продолжительности жизни, мировым демографическим кризисом и дискриминацией людей пожилого возраста, что находит отражение в современной художественной литературе. Целью статьи является изучение репрезентаций творческихличностей пожилого возраста в  сборнике комедий современной американской женщины-драматурга Тины Хау 1989 г. Динамика геронтогенеза представлена моделированием положительных образов, привлеченных к созерцанию или участию в креативных процессах, в трех пьесах. Выделены следующие геронтологические маркеры: фрагменты жизненного обозрения в форме воспоминаний, танатологический дискурс, рассуждения о суициде среди представителей поздней взрослости и реплики самостереотипизации. Установлена амбивалентная модель солидарности-конфликта в разработке сети отношений между стареющими персонажами и их взрослыми детьми