The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Exploration and structure-based engineering of alkenal double bond reductases catalyzing the CαCß double bond reduction of coniferaldehyde.

Lignin, a complex aromatic polymer, represents a significant obstacle in lignocellulosic biomass utilization. The polymerization of lignin occurs by radical couplings, which mainly form ether and C-C bonds between monolignol units. The chemical stability of these bonds between monolignol units causes the recalcitrant nature of lignin. Since the Cα-Cß double bond in the monolignols is a crucial chemical feature for the radical coupling, reduction of the double bond would decrease the degree of lignin polymerization, avoiding the recalcitrance of lignin. To develop a method of lignin engineering, we have focused on alkenal double bond reductases (DBR), which can reduce the Cα-Cß double bond of a monolignol precursor. Here, a novel bacterial DBR from Parvibaculum lavamentivorans DS-1 (PlDBR) was found. This enzyme can reduce the side-chain double bond of coniferaldehyde (CALD) and has a 41% amino-acid sequence identity with CALD DBR from Arabidopsis thaliana (AtDBR). The crystal structure of the PlDBR showed that it has a larger substrate-binding pocket than AtDBR, conferring broader substrate specificity on the former. Structural and mutation analyses of PlDBR and AtDBR suggested that Tyr51 and Try252 are critical residues for the catalytic activity of PlDBR. In addition, Tyr81 of AtDBR appears to cause substrate inhibition. Replacing Tyr81 of AtDBR with a smaller amino-acid residue, as in the AtDBR variants Tyr81Leu and Tyr81Ala, resulted in a substantially higher CALD-reducing activity compared to the wild type. These variants would be promising candidates for lignin manipulation to decrease the recalcitrance of lignocellulosic biomass.

Inhibition of activated K-opioid receptor to myocardical hypertrophy induced by ET-1 in neonatal rats by extracellular signal-regulated kinase pathway / 中国药理学通报

Aim To investigate the inhibitory effect of kappa-opioid receptor(κ-OR)stimulation on extracellular signal-regulated kinase pathway on ET-1-induced cardiomyocyte hypertrophy in vitro cultured myocardial cells from neonatal rats.Methods Myocardial cells of neonatal rats were cultured in vitro.The hypertrophic myocytes were induced by ET-1(10 nmol·L-1)before κ-OR agonist U50488H(1 μmol·L-1)was administered.The antihypertrophic effect of κ-OR stimulation was observed in the presence of U0126(1μmol·L-1), Ro-31-8220(50 nmol·L-1)and PTX(5 mg·L-1).The cardiomyocytes volume was measured by computer photographalysis system.The relative expression of ERK1/2 was determined by Western blot.The morphological changes in cardiomyocytes were observed under an inverted phase contrast microscope.The expression of mRNA of atrial natriuretic peptide(ANP)was determined by RT-PCR.Results Compared with normal control group, ET-1 could induced cardiomyocyte hypertrophy.Compared with ET-1 model group, U50488H(1 μmol·L-1)could obviously inhibit ET-1-induced increase of the cardiomyocytes volume, expression of ANPmRNA and expression of ERK1/2, which was similar to U0126(1 μmol·L-1)and Ro-31-8220(50 nmol·L-1); however, the inhibitory effects of U50488H were partly lost when preincubated with U0126(1 μmol·L-1)and Ro-31-8220(50 nmol·L-1); the inhibitory effects of U50488H, U0126(1 μmol·L-1)and Ro-31-8220(50 nmol·L-1)were lost when preincubated with NOR-BNI.Conclusion The stimulation of kappa-opiod can inhibit myocardial hypertrophy induced by ET-1, which is possibly via attenuating ERK1/2.

FAD-dependent C-glycoside-metabolizing enzymes in microorganisms: Screening, characterization, and crystal structure analysis.

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.

Characterization of a novel type of carbonic anhydrase that acts without metal cofactors.

BACKGROUND: Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, ß, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. RESULTS: Here, we report that a previously uncharacterized protein group, named "COG4337," constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+ß barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. CONCLUSIONS: COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Mailuoning Compound for Treatment of Early Steroid-induced Osteonecrosis of Femoral Head in Rats by Obturator Nerve Block / 中国实验方剂学杂志

Objective: To investigate the effect and mechanism of Mailuoning Compound for treatment of early steroid-induced osteonecrosis of femoral head (SONFH) in rats by obturator nerve block. Method: 24 rats were injected with endotoxin 10 μg·kg-1 through tail vein. After 24 hours, prednisolone acetate 20 mg·kg-1 was given by intraperitoneal injection, once every 24 hours for 3 consecutive days. After successful modeling, the rats were randomly divided into the model group (n=12), the treatment group (n=12) and the normal control group (n=6). In the treatment group, 2 mL·kg-1 of Mailuoning compound was injected into the obturator nerve from the 4th day, 3 times a week for 8 weeks. The arterial blood was collected from rats on the first day of the 9th week after model building to detect the content of blood lipid; the femoral head was taken to prepare the paraffin section, and the pathological changes of femoral head was observed and the changes of empty bone lacuna rate, bone trabecular area and bone lacuna area were quantitatively analyzed; The changes of bone morphogenetic proteins(BMPs),transforming growth factor-β1(TGF-β1),vascular endothelial cell growth factor(VEGF),and Ⅷ factor related antigen(Ⅷ-R Ag) were quantitatively analyzed by immunohistochemical method. Result: In the model group, the bone trabeculae were sparse, thin, disorganized and broken; some of the bone cells were necrotic and the number of empty bone lacunae was increased. In the treatment group, the number of trabeculae was increased; the structure was clear, most of which was normal bone cells, with a few necrotic bone cells, and the number of empty bone lacunae was decreased obviously. The rate of empty bone lacuna and the area of bone lacuna in the treatment group were significantly lower than those in the model group (Pβ1 and the microvessel density of Ⅷ-R Ag in the treatment group were significantly higher than those in the model group (PPConclusion: Mailuoning compound can improve the microcirculation state of femoral head, promote the formation of new bone and blood vessel in femoral head by regulating the expression of VEGF, BMPs, TGF-β1, Ⅷ-R Ag and down-regulating blood lipid content, thus effectively controlling the development of early SONFH. This can provide a theoretical basis for the treatment of early SONFH.

Y-27632 promotes differentiation of human embryonic stem cells into neuron-like cells / 中国组织工程研究

BACKGROUND: Y-27632 is a Rho-associated coil-formed protein kinase inhibitor that can regulate the self-renewal of stem cells, promote clonal formation and cell survival, and regulate and protect neuronal cell growth and development. How to improve the differentiation efficiency of embryonic stem cells into neuron-like cells is highly important for nerve injury repair and nerve regeneration. OBJECTIVE: To investigate the effect of Y-27632 on the differentiation efficiency and function of human embryonic stem cells into neuron-like cells. METHODS: After resuscitation, human embryonic stem cells at passage 16 were subjected to morphological observation and staining identification. The embryoid bodies were prepared by suspension culture, and after 8 days of incubation, the cells were cultured in Sato medium containing different concentrations of Y-27632 (0, 5, 10, 20, 40 μmol/L) for 10 days and identification by staining. After induction for 18 days, the differentiation efficiency and neurite outgrowth were identified by immunofluorescence staining. RESULTS AND CONCLUSION: The human embryonic stem cells co-expressed Oct4 and SSEA-3 stem cell specific markers. After suspension culture for 8 days and further adherent culture for 10 days, the cells could be differentiated into neuron-like cells with neurogenic morphology and expressing Tuj-1. Y-27632, especially at a concentration of 10 μmol/L, not only promoted cell proliferation (a significant increase in adherent cells an Tuj-1 positive cells), but also facilitated cell differentiation into neurons. Immunofluorescence staining findings showed that 10 μmol/L Y-27632 significantly increased the number of Tuj-1 positive cells and neurites and the length of neurites after 18 days of differentiation. These results indicate that Y-27632 not only promotes the differentiation of human embryonic stem cells into neuron-like cells, but also accelerates neurite outgrowth.

The crystal structure of a new O-demethylase from Sphingobium sp. strain SYK-6.

In the cell, tetrahydrofolate (H4 folate) derivatives with a C1 unit are utilized in various ways, such as for the synthesis of amino acids and nucleic acids. While H4 folate derivatives with the C1 unit are typically produced in the glycine cleavage system, Sphingobium sp. strain SYK-6, which can utilize lignin-derived aromatic compounds as a sole source of carbon and energy, lacks this pathway, probably due to its unique nutrient requirements. In this bacterium, H4 folate-dependent O-demethylases in catabolic pathways for lignin-derived aromatic compounds seem to be involved in the C1 metabolism. LigM is one of the O-demethylases and catalyzes a C1-unit transfer from vanillate (VNL) to H4 folate. As the primary structure of LigM shows a similarity to T-protein in the glycine cleavage system, we hypothesized that LigM has evolved from T-protein, acquiring its unique biochemical and biological functions. To prove this hypothesis, structure-based understanding of its catalytic reaction is essential. Here, we determined the crystal structure of LigM in apo form and in complex with substrates and H4 folate. These crystal structures showed that the overall structure of LigM is similar to T-protein, but LigM has a few distinct characteristics, particularly in the active site. Structure-based mutational analysis revealed that His60 and Tyr247, which are not conserved in T-protein, are essential to the catalytic activity of LigM and their interactions with the oxygen atom in the methoxy group of VNL seem to facilitate a methyl moiety (C1-unit) transfer to H4 folate. Taken together, our structural data suggest that LigM has evolved divergently from T-protein. DATABASES: All atomic coordinates of the crystal structures determined in this study have been deposited to PDB. LigM: 5X1I, LigM-VNL complex: 5X1J, LigM-3-O-methylgallate complex: 5X1K, LigM-H4 folate complex: 5X1IL, LigM-H4 folate-protocatechuate (PCA) complex (P21 21 2): 5X1M, LigM-H4 folate-PCA complex (P31 21): 5X1N.

Inhibition on Numb/Notch signal pathway enhances radiosensitivity of lung cancer cell line H358.

The objective of the study is to investigate the effects of the Numb/Notch signal pathway on the radiosensitivity of lung cancer cell line H358. MTT assay and colony forming assay were used to detect the effects of different doses of X-rays and MW167 on the in vitro proliferation of the lung cancer cell line H358. Flow cytometry was applied to evaluate the effects of X rays on the apoptosis of H358. Scratch assay and Transwell invasion assay were used to examine the effects of X-rays on the migration and invasion abilities of H358. The mRNA and protein expressions in the signal pathway were detected by real-time PCR and western blot. Assays in vitro confirmed the effects of the Numb/Notch pathway inhibitor on the radiosensitivity to lung cancer. MW167 enhanced the inhibiting effects of X-ray on the proliferation of H358 cell line. After the addition of MW167, the apoptosis rates significantly increased, but the invasion and migration abilities decreased significantly. Meanwhile, MW167 could dose-dependently promote the increase of expression of Numb, which is the upstream gene of the Numb/Notch signaling pathway, but inhibit the expression of and HES1. In vivo experiments revealed that cell proliferation was suppressed in the radiation, pathway inhibitor, and pathway inhibitor + radiation groups, and the pathway inhibitor + radiation group exhibited more active anti-tumor ability when compared with the blank group (all P < 0.05); Numb expression was up-regulated, but Notch1 and HES1 expressions were down-regulated in those three groups, and also, the pathway inhibitor + radiation group exhibited more significant alternation when compared with the blank group (all P < 0.05); cell apoptosis was promoted in those three groups, and the pathway inhibitor + radiation group showed more active apoptosis when compared with the blank group (all P < 0.05). Repression of the Numb/Notch pathway enhances the effects of radiotherapy on the radiosensitivity of the lung cancer cell line H358, and thus the Numb/Notch pathway may be a new target of radiotherapy for lung cancer.

Nimotuzumab enhances the radiosensitivity of cancer cells in vitro by inhibiting radiation-induced DNA damage repair.

BACKGROUND: Nimotuzumab is a humanized IgG1 monoclonal antibody specifically targeting EGFR. In this study, we aimed to investigate the molecular mechanisms of nimotuzumab in its effects of enhancing cancer cell radiosensitivity. PRINCIPAL FINDING: Lung cancer A549 cells and breast cancer MCF-7 cells were pretreated with or without nimotuzumab for 24 h before radiation to perform the clonogenic survival assay and to analyze the cell apoptosis by flow ctyometry. γ-H2AX foci were detected by confocal microscopy to assess the effect of nimotuzumab on radiation induced DNA repair. EGFR activation was examined and the levels of DNA damage repair related proteins in A549 cells at different time point and at varying doses exposure after nimotuzumab and radiation treatment were examined by Western blot. Pretreatment with nimotuzumab reduced clonogenic survival after radiation, inhibited radiation-induced EGFR activation and increased the radiation-induced apoptosis in both A549 cells and MCF-7 cells. The foci of γ-H2AX 24 h after radiation significantly increased in nimotuzumab pretreated cells with different doses. The phosphorylation of AKT and DNA-PKcs were remarkably inhibited in the combination group at each dose point as well as time point. CONCLUSIONS: Our results revealed that the possible mechanism of nimotuzumab enhancing the cancer radiosensitivity is that nimotuzumab inhibited the radiation-induced activation of DNA-PKcs through blocking the PI3K/AKT pathway, which ultimately affected the DNA DSBs repair.

Stimulation of adenosine A1 receptor attenuates angiotensin II induced myocardial hypertrophy in neonatal rats via the extracellular signal-regulated kinase signal pathways / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the impact of adenosine A1 receptor stimulation on extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathways on angiotensin II (AngII) stimulated cardiomyocytes of neonatal rats in vitro.</p><p><b>METHODS</b>Cardiomyocytes of neonatal rats were cultured in vitro. Cardiomyocytes hypertrophy was induced by AngII (0.1 µmol/L). The antihypertrophic effect of adenosine A1 receptor stimulation via adenosine A1 receptor agonist R-PIA (1 µmol/L) was observed in the presence or absence of ERK1/2 inhibitor 1, 4-Diamino-2, 3-dicyano-1, 4-bis(o-aminophenylmercapto) butadiene (U0126) 1 µmol/L, PKC inhibitor Ro-31-8220 (50 nmol/L), and pertussis toxin (PTX, 5 mg/L). The total protein content was assayed by the method of Lowry. The expression of mRNA of atrial natriuretic peptide (ANP) was determined by RT-PCR. [Ca(2+)]i was measured by confocal microscope using Fluo-3/AM as fluorescent indicator. The relative expression of ERK1/2 was determined by Western blot.</p><p><b>RESULTS</b>Compared with normal control group, AngII induced significant cardiomyocyte hypertrophy. Compared with AngII group, R-PIA significantly inhibited AngII-induced increase of the protein content, cardiomyocytes volume and expression of ERK1/2, calcium ion fluorescence intensity, similar as U0126 and Ro-31-8220. The inhibitory effects on AngII induced cardiomyocytes hypertrophy of R-PIA were lost when preincubated with PTX.</p><p><b>CONCLUSION</b>Adenosine A1 receptor can inhibit AngII induced cardiomyocyte hypertrophy through downregulating ERK signal pathways and reducing intracellular Ca(2+).</p>

Effects and Mechanism of Adenosine A_1 Receptor Agonist on Isoproterenol Induced Hypertrophy in Cultured Neonatal Rats Myocardial Cells / 中华高血压杂志

Objective To demonstrate the inhibitory effect of adenosine A_1 receptor agonist R(-)-N6-(2-phe- nylisopropyl) adenosine (R-HA) and cross-talk between adenosine A_1 receptor and CaMKII on Isoproterenol (Iso)- induced hypertrophy in cultured myocardial cells in neonatal rats.Methods The protein synthesis was determined by incorporation of [~3H]-leucine into myocyte protein.The expression of CaMK Ⅱ ? B was determined by Western- blot.The [Ca~(2+)]i transient was measured in myocytes loaded with fura-2 by the spectrofluorometric method. Results R-PIA (1?mol/L) inhibited Iso(10 ?mol/L)-induced increase of [~3 H-]-leucine incorporation [(R-PIA: 974.8?58.6) vs (Iso:1220.8?240.5) count per min per well,P

Inhibitory effects of kappa-opioid receptor stimulation on cultured myocardial cells / 中国应用生理学杂志

<p><b>AIM</b>In order to study the effects of kappa-opioid receptor activation, we used cultured cardiomyocytes to study the inhibitory effects of U50,488H on cellular proliferation, protein content in the presence or absence of nor-binaltorphimine (nor-BNI).</p><p><b>METHODS</b>The cellular proliferation was determined with crystal violet staining and the protein content was assayed with Lowry's method.</p><p><b>RESULTS</b>A kappa-opioid receptor agonist U50,488H at 0.1 micromol/L-10 micromol/L inhibited the cellular proliferation and protein content of cultured myocardial cells in a dose-dependent manner. The inhibitory effects of U50,488H were completely blocked by pretreatment with nor-BNI, a specific kappa-opioid receptor antagonist at 1 micromol/L.</p><p><b>CONCLUSION</b>U50,488H inhibited the cultured myocardial cells' growth. The inhibitory effects of U50,488H are involved in mediating the action of kappa-opioid receptor stimulation.</p>