The reaction of hemin with H2O2

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.     

Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2

Disabled-2 (Dab2) is a widely expressed relative of Disabled-1, a neuron-specific signal-transduction protein that binds to and receives signals from members of the low-density lipoprotein receptor (LDLR) family. Members of the LDLR family internalize through clathrin-coated pits and vesicles to endosomes, from where they return to the cell surface through the secretory pathway. In this study, we show that the Dab2 phosphotyrosine-binding domain binds peptides containing the sequence FXNPXY. This core sequence is found in the intracellular domains of LDLR family members and is important for receptor internalization. Dab2 transiently colocalizes with the LDLR in clathrin-coated pits, but is absent from endosomes and lysosomes. Dab2 is alternatively spliced and its localization depends on a region of the protein that contains two DPF motifs that are present in the p96 Dab2 protein and absent in the p67 splice variant. This region is sufficient to confer Dab2 binding to the α-adaptin subunit of the clathrin adaptor protein, AP-2. Overexpression of p96 but not of p67 Dab2 disrupts the localization of AP-2. These findings suggest that in addition to previously reported signal-transduction functions, Dab2 could also act as an adaptor protein that may regulate protein trafficking.     

Interaction of Shc with Grb2 regulates association of Grb2 with mSOS.

The adapter protein Shc has been implicated in Ras signaling via many receptors, including the T-cell antigen receptor (TCR), B-cell antigen receptor, interleukin-2 receptor, interleukin-3 receptor, erythropoietin receptor, and insulin receptor. Moreover, transformation via polyomavirus middle T antigen is dependent on its interaction with Shc and Shc tyrosine phosphorylation. One of the mechanisms of TCR-mediated, tyrosine kinase-dependent Ras activation involves the simultaneous interaction of phosphorylated Shc with the TCR zeta chain and with a second adapter protein, Grb2. Grb2, in turn, interacts with the Ras guanine nucleotide exchange factor mSOS, thereby leading to Ras activation. Although it has been reported that in fibroblasts Grb2 and mSOS constitutively associate with each other and that growth factor stimulation does not alter the levels of Grb2:mSOS association, we show here that TCR stimulation leads to a significant increase in the levels of Grb2 associated with mSOS. This enhanced Grb2:mSOS association, which occurs through an SH3-proline-rich sequence interaction, is regulated through the SH2 domain of Grb2. The following observations support a role for Shc in regulating the Grb2:mSOS association: (i) a phosphopeptide corresponding to the sequence surrounding Tyr-317 of Shc, which displaces Shc from Grb2, abolished the enhanced association between Grb2 and mSOS; and (ii) addition of phosphorylated Shc to unactivated T cell lysates was sufficient to enhance the interaction of Grb2 with mSOS. Furthermore, using fusion proteins encoding different domains of Shc, we show that the collagen homology domain of Shc (which includes the Tyr-317 site) can mediate this effect. Thus, the Shc-mediated regulation of Grb2:mSOS association may provide a means for controlling the extent of Ras activation following receptor stimulation.     

Template-directed synthesis with 2-aminoadenosine

Summary A random copolymer, poly(CA), containing approximately equal amounts of cytidine (C) and adenosine (A), when incubated with a mixture of guanosine-5-phosphoro-(2-methylimidazole) (2-MeImpG) and uridine-5-phosphoro-(2-methylimidazole) (2-MeImpU), facilitates the incorporation of uridine (U) into oligomeric products with low efficiency. If 2-aminoadenosine (aA) is substituted for adenosine in the template, U is incorporated into the products with much higher efficiency. Random copolymers of C and U act as templates for the efficient synthesis of oligomers from 2-MeImpG and 2-MeImpA only if the concentration of substrates is relatively high (0.1 M). The substitution of 2-MeImpaA permits the reaction to occur with much lower substrate concentrations. This effect is most prominent for template containing large amounts of U.     

Real time with Caspase-2

Comment on: Bouchier-Hayes L, et al. Mol Cell 2009;35:830-40.     

Transthyretin interacts with metallothionein 2

Transthyretin (TTR) is a 55 kDa homotetrameric protein known for the transport of thyroxine and the indirect transportation of retinol. Within the central nervous system, TTR is primary synthesized and secreted into the cerebral spinal fluid by the choroid plexus (CP), whereas most TTR in the systemic circulation is produced and secreted by the liver. TTR is involved in two types of amyloid disease, the senile systemic amyloidosis and the familial amyloidotic polyneuropathy. TTR has also been implicated in the sequestration of amyloid beta peptide (Abeta), preventing its deposition. To explore other biological roles for TTR, we searched for protein-protein interactions using the yeast two-hybrid system with the full-length human TTR cDNA as bait. We found a novel interaction between TTR and metallothionein 2 (MT2) in human liver. This interaction was confirmed by competition binding assays, co-immunoprecipitation, cross-linking, and Western blotting experiments. Binding studies using MT1 showed a saturable specific interaction with TTR with a Kd of 244.8 +/- 44.1 nM. Western blotting experiments revealed a TTR-MT1/2 protein complex present in rat CP and kidney tissue extracts. Immunofluorescence experiments, in CP primary cell cultures and in CP paraffin sections, showed co-localization of TTR and MT1/2 in the cytoplasm of epithelial CP cells and localization of MT1/2 in the endoplasmic reticulum. Moreover, dot blot immunoassays of rat CSF provided the first evidence, to our knowledge, of circulating metallothionein in CSF. Taken together, we suggest that TTR-MT1/2 complexes may be functionally significant not only in healthy conditions but also in Abeta deposition in Alzheimer disease, thereby providing a novel potential therapeutic target.     

Side products of glycosidation with selected 2-acetamido-2-deoxy-D-glucopyranosides

Allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-formyl-alpha-D-glucopyranoside, N-acetyl-2,3,4-tri-O-acetyl-L-fucopyranosylamine and products of O-acetyl group migration were found as side products during glycosidation of selected 2-acetamido-2-deoxy-D-glucopyranosides.     

Menstrual induction with PGF2 alpha and PGE2.

The "prostaglandin impact" (PGI), a massive intrauterine dose of PG, converts the refractory pregnant uterus into a reactive organ by provoking a regulatory imbalance. This regulatory conversion releases the endogenous mechanism of menstruation or abortion. During initial studies, PGI successfully provoked menstrual induction (MI) in 22 and subsequently in 65 volunteers. These results were confirmed and complemented by 2 independent trials in 14 and 36 gravidas respectively. The best clinical outcome was obtained in 20 volunteers, when a "PG-Pellet" (a mini-suppositorium) was inserted in utero, containing only 2.5 mg PGF2alpha. These 157 trials in sedated volunteers had the common features of over 90% efficiency, transient and medically acceptable side effects and infrequent complications. The present study of 542 volunteers focused upon the collection of clinical data regarding efficacy, side effects and complications of MI. All patients had committee approval for legal abortion, during the 2nd week of their missed menstrual period. They volunteered to participate because of their preference for pharmacological rather than surgical pregnancy termination. The clinical outcome of the 542 MI with 5 mg PGF2alpha (428 cases) and 1.5 mg PGE2 (114 cases) was identical. On the average, 95% of the gravidas had complete evacuation of the uterus with the clinical symptoms of delayed menstruation rather than abortion; they experienced spontaneous menstruation in 34 days after having received a single dose of PG.     

5'-Esters of 2'-deoxyadenosine and 2-chloro-2'-deoxyadenosine with cell differentiation-provoking agents

Phenylacetic and retinoic acids are carboxyacidic cell differentiating agents displaying anticancer activities. We report on a new class of compounds including the 5'-esters of 2'-deoxyadenosine (dA) or 2-chloro-2'-deoxyadenosine (cladribine, 2CdA) and the aforementioned acids. The rationale behind the synthesis of these esters was that if they are hydrolyzed inside the lymphoid cells, either dA will be removed from the intracellular environment by deamination, or 2CdA will be phosphorylated and accumulated. In either case targetted delivery of the differentiating agent to the lymphoid cells may be envisaged. The said compounds were synthesized by the Mitsunobu procedure employing triphenylphosphine and azadicarboxylic acid esters, and their stability was tested against various esterases. Esters of dA and 2CdA with phenylacetic acids were found to be resistant to enzymatic hydrolysis, whereas those with retinoic acids were efficiently hydrolyzed by commercially available hepatic esterase as well as by esterases present in the blood plasma and in diluted human lymphocyte lysate. Susceptibility to enzymatic hydrolysis was found to be a prerequisite of cytotoxic and/or differentiating activity of these esters in leukemic cell lines.     

抵抗素与肥胖和2型糖尿病

抵抗素(resistin)是近年来新发现的一个由脂肪组织特异表达分泌的细胞因子,其在前脂肪细胞分化过程中抑制脂肪生成.众多的研究显示抵抗素可诱导脂肪、肝脏及肌肉组织产生胰岛素抵抗,损伤机体的糖脂代谢功能.由于胰岛素抵抗在一些其他代谢性疾病及并发症如动脉粥样硬化及高血压发病机制中也发挥重要作用,提示抵抗素有可能介入了这些疾病的发病过程.本文简要介绍抵抗素的结构、分布及表达调控,并重点分析抵抗素在胰岛素抵抗中的作用.     

抵抗素与肥胖和2型糖尿病

抵抗素（resistin）是近年来新发现的一个由脂肪组织特异表达分泌的细胞因子,其在前脂肪细胞分化过程中抑制脂肪生成。众多的研究显示抵抗素可诱导脂肪、肝脏及肌肉组织产生胰岛素抵抗,损伤机体的糖脂代谢功能。由于胰岛素抵抗在一些其他代谢性疾病及并发症如动脉粥样硬化及高血压发病机制中也发挥重要作用,提示抵抗素有可能介入了这些疾病的发病过程。本文简要介绍抵抗素的结构、分布及表达调控,并重点分析抵抗素在胰岛素抵抗中的作用。     

Residue-level determinants of angiopoietin-2 interactions with its receptor Tie2

We combined computational and experimental methods to interrogate the binding determinants of angiopoietin-2 (Ang2) to its receptor tyrosine kinase (RTK) Tie2—a central signaling system in angiogenesis, inflammation, and tumorigenesis. We used physics-based electrostatic and surface-area calculations to identify the subset of interfacial Ang2 and Tie2 residues that can affect binding directly. Using random and site-directed mutagenesis and yeast surface display (YSD), we validated these predictions and identified additional Ang2 positions that affected receptor binding. We then used burial-based calculations to classify the larger set of Ang2 residues that are buried in the Ang2 core, whose mutations can perturb the Ang2 structure and thereby affect interactions with Tie2 indirectly. Our analysis showed that the Ang2-Tie2 interface is dominated by nonpolar contributions, with only three Ang2 and two Tie2 residues that contribute electrostatically to intermolecular interactions. Individual interfacial residues contributed only moderately to binding, suggesting that engineering of this interface will require multiple mutations to reach major effects. Conversely, substitutions in substantially buried Ang2 residues were more prevalent in our experimental screen, reduced binding substantially, and are therefore more likely to have a deleterious effect that might contribute to oncogenesis. Computational analysis of additional RTK-ligand complexes, c-Kit-SCF and M-CSF-c-FMS, and comparison to previous YSD results, further show the utility of our combined methodology.