Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC

The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the “Y–T coupling” mechanism, wherein the χ₁ rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC^R), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ₁ rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC^R in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted “Y–T coupling” mechanism of activation in NtrC^R.     

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG)

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.     

The role of the abnormalities in the distal pathway of cholesterol biosynthesis in the Conradi–Hünermann–Happle syndrome

Conradi–Hünermann–Happle syndrome (CDPX2, OMIM 302960) is an inherited X-linked dominant variant of chondrodysplasia punctata (CP) caused by mutations in one gene of the distal pathway of cholesterol biosynthesis. It exhibits intense phenotypic variation and primarily affects the skin, bones and eyes. The ichthyosis following Blaschko's lines, chondrodysplasia punctata and cataracts are the typical clinical findings. The cardinal biochemical features are an increase in 8(9)-cholestenol and 8-dehydrocholesterol (8DHC), which suggest a deficiency in 3β-hydroxysteroid-Δ8,Δ7-isomerase, also called emopamil binding protein (EBP). The EBP gene is located on the short arm of the X chromosome (Xp11.22–p11.23) and encodes a 230 amino acid protein with dual function. Explaining the clinical phenotype in CDPX2 implies an understanding of both the genetics and biochemical features of this disease. CDPX2 displays an X-linked dominant pattern of inheritance, which is responsible for the distribution of lesions in some tissues. The clinical phenotype in CDPX2 results directly from impairment in cholesterol biosynthesis, and indirectly from abnormalities in the hedgehog signaling protein pathways. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Charge ordering in the metal–insulator transition of V-doped CrO<Subscript>2</Subscript> in the rutile structure

Electronic, magnetic, and structural properties of pure and V-doped CrO₂ were extensively investigated utilizing density functional theory. Usually, pure CrO₂ is a half-metallic ferromagnet with conductive spin majority species and insulating spin minority species. This system remains in its half-metallic ferromagnetic phase even at 50% V-substitution for Cr within the crystal. The V-substituted compound Cr_0.5V_0.5O₂ encounters metal–insulator transition upon the application of on-site Coulomb repulsion U?=?7 eV preserving its ferromagnetism in the insulating phase. It is revealed in this study that Cr³⁺-V⁵⁺ charge ordering accompanied by the transfer of the single V-3d electron to the Cr-3dt_2g orbitals triggers metal–insulator transition in Cr_0.5V_0.5O₂. The ferromagnetism of Cr_0.5V_0.5O₂ in the insulating phase arises predominantly due to strong Hund’s coupling between the occupied electrons in the Cr-t_2g states. Besides this, the ferromagnetic Curie temperature (T_c) decreases significantly due to V-substitution. Interestingly, a structural distortion is observed due to tilting of CrO₆ or VO₆ octahedra across the metal–insulator transition of Cr_0.5V_0.5O₂.

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Graphical abstract The V-doped compound Cr_0.5V_0.5O₂ is found a half-metallic ferromagnet (HMF) in the absence of on-site Coulomb interaction (U). This HMF behavor maintains up to U?=?6 eV. Eventually, this system encounters metal-insulator transition (MIT) upon the application of U?=?7 eV with a band gap of E_g ~ 0.31 eV. Nevertheless, applications of higher U widen the band gaps. In this figure, calculated total (black), Cr-3d (red), V-3d (violet), and O-2p (blue) DOS of Cr_0.5V_0.5O₂ for U?=?8 eV are illustrated. The system is insulating with a band gap of E_g ~ 0.7 eV.

     

Disruption to the 5-HT<Subscript>7</Subscript> Receptor Following Hypoxia–Ischemia in the Immature Rodent Brain

It has become increasingly evident the serotonergic (5-hydroxytryptamine, 5-HT) system is an important central neuronal network disrupted following neonatal hypoxic–ischemic (HI) insults. Serotonin acts via a variety of receptor subtypes that are differentially associated with behavioural and cognitive mechanisms. The 5-HT₇ receptor is purported to play a key role in epilepsy, anxiety, learning and memory and neuropsychiatric disorders. Furthermore, the 5-HT₇ receptor is highly localized in brain regions damaged following neonatal HI insults. Utilising our well-established neonatal HI model in the postnatal day 3 (P3) rat pup we demonstrated a significant decrease in levels of the 5-HT₇ protein in the frontal cortex, thalamus and brainstem one week after insult. We also observed a relative decrease in both the cytosolic and membrane fractions of 5-HT₇. The 5-HT₇ receptor was detected on neurons throughout the cortex and thalamus, and 5-HT cell bodies in the brainstem. However we found no evidence of 5-HT₇ co-localisation on microglia or astrocytes. Moreover, minocycline treatment did not significantly prevent the HI-induced reductions in 5-HT₇. In conclusion, neonatal HI injury caused significant disruption to 5-HT₇ receptors in the forebrain and brainstem. Yet the use of minocycline to inhibit activated microglia, did not prevent the HI-induced changes in 5-HT₇ expression.     

Sympatric speciation of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Monohostal specialization in the host–parasite marmot–flea (<Emphasis Type="Italic">Marmota sibirica–Oropsylla silantiewi</Emphasis>) system

An ecological scenario of the origin of the plague microbe that is interpreted in the light of modern Darwinism (synthetic theory of evolution) is presented. It is shown that the plague microbe emerged from a clone of the psychrophilic saprozoonotic pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan time, 22000–15000 years ago, in the monohostal Mongolian marmot (Marmota sibirica)–flea (Oropsylla silantiewi) host–parasite system. It was noted that the evolutionary process described corresponds to the sympatric form of speciation by transition of the clone of migrant founders to a new, already-existing ecological niche. It was established that monohostal specialization of the plague microbe was made possible due to heterothermia (5–37°C) of marmots in the hibernation period. The factors of the speciation process—isolation, the struggle for existence, and natural selection—were analyzed.     

Structure of the Mn<Subscript>4</Subscript>–Ca cluster as derived from X-ray diffraction

The catalytic centre for light-induced water oxidation in photosystem II (PSII) is a multinuclear metal cluster containing four manganese and one calcium cations. Knowing the structure of this biological catalyst is of utmost importance for unravelling the mechanism of water oxidation in photosynthesis. In this review we describe the current state of the X-ray structure determination at 3.0 A resolution of the water oxidation complex (WOC) of PSII. The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and possible perspectives for the future are outlined, including the combination of X-ray diffraction and X-ray spectroscopy on single crystals.     

A Phosphothreonine Residue at the C-Terminal End of the Plasma Membrane H+-ATPase Is Protected by Fusicoccin-Induced 14–3–3 Binding

We have isolated the plasma membrane H⁺−ATPase in a phosphorylated form from spinach (Spinacia oleracea L.) leaf tissue incubated with fusicoccin, a fungal toxin that induces irreversible binding of 14–3–3 protein to the C terminus of the H⁺-ATPase, thus activating H⁺ pumping. We have identified threonine-948, the second residue from the C-terminal end of the H⁺-ATPase, as the phosphorylated amino acid. Turnover of the phosphate group of phosphothreonine-948 was inhibited by 14–3–3 binding, suggesting that this residue may form part of a binding motif for 14–3–3. This is the first identification to our knowledge of an in vivo phosphorylation site in the plant plasma membrane H⁺-ATPase.     

The Effects of FeCl<Subscript>3</Subscript> and Fe–EDTA on the Development of Psoriasis

The effects of FeCl₃ and Fe–EDTA on the development of psoriasis were studied in the mouse model of vaginal epithelium and tail epidermis. The mitoses of vaginal epithelial cell in female mice of their estrogenic stage and the formation of granular cell layers in male mouse tail scale were observed. Mice were randomly divided into eight groups and treated with normal saline, methotrexate, and different doses of two iron forms, FeCl₃ and Fe–EDTA, respectively, for 10 days. To explore the influence of FeCl₃ and Fe–EDTA on the excretion of Cu, Fe, Zn, Ca, Mg, Mn, and Se, the concentration of those elements in liver and kidney was analyzed by atomic absorption spectrometry. The different doses of FeCl₃ or Fe–EDTA could obviously inhibit the mitoses of vaginal epithelial cell (p< 0.05) and promote the formation of granular cell layers in mice tail scale (p < 0.05). No statistically significant results were found between the groups of FeCl₃ and Fe–EDTA, and between experimental groups and methotrexate group acted as the positive control (p>0.05). Compared with the negative group, the concentrations of Cu, Fe, Zn, Ca, Mg, Mn, and Se in liver and kidney of experimental groups and positive control group were not significantly changed (p > 0.05). FeCl₃ and Fe–EDTA are as effective as methotrexate on inhibiting hyperplasia of epidermal cells and increasing the formation of granular cell layers, and the concentrations of Cu, Fe, Zn, Ca, Mg, Mn, and Se in liver and kidney of experimental groups and positive control group were not significantly changed compared with the negative group, possibly retarding the development of psoriasis.     

Effects of Host Interspecific Interaction in the <Emphasis Type="Italic">Maculinea</Emphasis>–<Emphasis Type="Italic">Myrmica</Emphasis> Parasite–Host System

A model of interspecific host competition in a system with one parasite (butterfly—Maculinea) and multiple potential hosts (ants—Myrmica) is presented. Results indicate that host interspecific competition increases the occurrence of multiple host behaviour in Maculinea natural populations but decreases the ability of the parasite populations to adapt to the most abundant host species. These qualitative predictions were compared with data on host specificity, with good agreement. Analysis of the data also indicates that Maculinea teleius and Maculinea arion respond differently to changes in relative host abundances. Maculinea teleius shows a larger fraction of sites where it displays multiple host behaviour and a larger fraction of sites where the niches of the hosts overlap. In some instances, Maculinea teleius is adapted to Myrmica hosts that are present in lower frequencies. Maculinea arion is locally more host-specific and occurs at sites where host interspecific competition is unlikely and is more frequently adapted to the most abundant host species.     

How uniform is the peptide plane geometry? A high-accuracy NMR study of dipolar C<Superscript>α</Superscript>–C′/H<Superscript>N</Superscript>–N cross-correlated relaxation

Highly precise and accurate measurements of very small NMR cross-correlated relaxation rates, namely those between protein H_i^N–N_i and C_i−1^α–C_i−1′ dipoles, are demonstrated with an error of 0.03 s⁻¹ for GB3. Because the projection angles between the two dipole vectors are very close to the magic angle the rates range only from −0.2 to +0.2 s⁻¹. Small changes of the average vector orientations have a dramatic impact on the relative values. The rates suggest deviation from idealized peptide plane geometry caused by twists around the C′–N bonds and/or pyramidalization of the nitrogen atoms. A clear alternating pattern along the sequence is observed in β strands 1, 3 and 4 of GB3, where the side chains of almost all residues with large positive rates are solvent exposed. In the α helix all rates are relatively large and positive. Some of the currently most accurate structures of GB3 determined by both high resolution X-ray crystallography and NMR are in satisfactory agreement with the experimental rates in the helix and β strand 3, but not in the loops and the two central strands of the sheet for which no alternating pattern is predicted.     

“RAF” neighborhood: Protein–protein interaction in the Raf/Mek/Erk pathway

The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade, however, the identification of unexpected in vivo functions of pathway components, as well as the discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to understand their mechanism of action, have boosted interest tremendously. From this large body of work, protein–protein interaction has emerged as a recurrent, crucial theme. This review focuses on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk with other signaling cascades. Mapping these interactions and finding a way of exploiting them for therapeutic purposes is one of the challenges of future molecule-targeted therapy.     

Anionic Selectivity Sequence of the Cl<Superscript>−</Superscript>–H<Superscript>+</Superscript> Symporter in the Synaptosomal Preparation from Rat Brain Cortex

The Na⁺/H⁺ exchanger has been the only unequivocally demonstrated H⁺-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl⁻–H⁺ symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl⁻–H⁺ symporter is NO₃⁻ > Br⁻ > Cl⁻ >> I⁻ = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 μM inhibits the gluconate-dependent alkalinization by 30 ± 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization. Special issue article in honor of Dr. Ricardo Tapia.     

On the First Reliable Record of the Ichthyosaur <Emphasis Type="Italic">Ophthalmosaurus icenicus</Emphasis> Seeley in the Oxfordian–Kimmeridgian Beds of European Russia

An incomplete skeleton of the ichthyosaur Ophthalmosaurus icenicus Seeley, 1874 excavated in the Oxfordian–Kimmeridgian beds south of the town of Syzran (Samara Region) is described. Members of the genus Ophthalmosaurus are characterized by a unique combination of characters, most of which are well-pronounced in the specimen described here: the extracondylar area of the basioccipital is reduced, but still visible beyond the occipital condyle and has a well-pronounced ventral notch; the scapular shaft is mediolaterelly flattened; the humerus has three distal facets: the anterior facet is the smallest; the facet for the accessory preaxial element has a sharpened anterior margin; the facet for the radius faces distally; the facet for the ulna faces posterodistally; the posterior margin of the ulna is concave and sharpened, involved in the perichondral ossification; the intermedium is rhomboid, with two distal facets equal in size for the second and third distal carpals; the epipodial and autopodial elements are round and thickened, loosely arranged in the fin. This specimen is the first reliable evidence of the presence of Ophthalmosaurus in the Oxfordian–Kimmeridgian of Russia. The previously described remains of a Kimmeridgian ichthyosaur referred to as O. undorensis Efimov, 1991 should be identified as Ophthalmosauridae indet.     

Translational biomaterials — the journey from the bench to the market — think ‘product’

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Protease-catalyzed synthesis of the tripeptide CCK<Subscript>26–28</Subscript>, a fragment of CCK-8

Summary. Two enzymatically synthetic strategies of the tripeptide derivative PhAc-Asp(OMe)-Tyr-Met-OAl are reported. The second strategy gains the advantage of more economical starting materials, less reaction steps and a higher overall isolated yield of this tripeptide fragment over the first strategy. The effect of the acyl-donor ester concentration and structure, the C-α protecting group of the nucleophile, reaction media, enzyme and the carrier on the tripeptide derivative synthesis were studied. This tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a potential therapeutic agent in the control of gastrointestinal function and also a drug candidate for the treatment of epilepsy.     

Effect of the β-amyloid peptide Aβ<Subscript>25–35</Subscript> and fullerene C<Subscript>60</Subscript> on the activity of enzymes in erythrocytes

The effect of the β-amyloid peptide Aβ_25–35 and fullerene C₆₀ on the activity of the cytoplasmic enzymes lactate dehydrogenase (LDH) and glutathione peroxidase (GLP), and membrane-bound phosphofructokinase (PFK) and Na⁺,K⁺-ATPase in human erythrocytes has been studied. When used in combination, the cytotoxins decrease the activity of LDH and PFK in a nonadditive manner; in this case, Aβ_25–35 protects PFK against the inhibitory effect of C₆₀. The activity of LDH, GLP, and PFK decreases within the first 2–20 min of incubation of erythrocytes with Aβ_25–35 in the absence of glucose. The addition of glucose sharply decreases the inhibitory action of Aβ_25–35 on LDH and GLP but does not affect the fourfold decrease in activity of PFK; the activity of membrane-bound Na⁺,K⁺-ATPase does not depend on the presence of glucose. Possible mechanisms of interaction of Aβ_25–35 and fullerene C₆₀ with the erythrocyte membrane and enzymes are discussed.     

Beyond the Boss and the Boys: Women and the Division of Labor in <Emphasis Type="Italic">Drosophila</Emphasis> Genetics in the United States, 1934–1970

The vast network of Drosophila geneticists spawned by Thomas Hunt Morgan’s fly room in the early 20th century has justifiably received a significant amount of scholarly attention. However, most accounts of the history of Drosophila genetics focus heavily on the “boss and the boys,” rather than the many other laboratory groups which also included large numbers of women. Using demographic information extracted from the Drosophila Information Service directories from 1934 to 1970, we offer a profile of the gendered division of labor within Drosophila genetics in the United States during the middle decades of the 20th century. Our analysis of the gendered division of labor supports a reconsideration of laboratory practices as different forms of work.