Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Interaction of recombinant analogs of spider silk proteins 1F9 and 2E12 with phospholipid membranes

Recombinant analogs of spider dragline silk proteins 1F9 and 2E12 are characterized by numerous repeats consisting of hydrophobic poly-Ala blocks and Gly-rich sequences with a substantial number of positively charged amino acid residues which suggest a pronounced ability to interact with negatively charged phospholipid membranes. Actually both proteins displayed substantial binding affinity towards lipid vesicles formed of acidic lipids as measured by fluorescence correlation spectroscopy (FCS) using rhodamine-labeled conjugates of the proteins. Both proteins did not induce liposome leakage, fusion or breakdown, but were able to bring about liposome aggregation. 1F9 was more active in the induction of liposome aggregation compared to 2E12. Interestingly, 2E12 markedly decreased the rate of calcium-induced liposome fusion. Circular dichroism data showed that binding of the proteins to negatively charged phosphatidylserine liposomes provoked transition from the left-handed helix of polyproline II (PPII) type to β-structures and α-helices. The data suggested predominantly surface location of membrane bound proteins without significant perturbation of their hydrophobic core.     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Cdh1, a Substrate-recruiting Component of Anaphase-promoting Complex/Cyclosome (APC/C) Ubiquitin E3 Ligase,Specifically Interacts with Phosphatase and Tensin Homolog (PTEN) and Promotes Its Removal from Chromatin

A pool of PTEN localizes to the nucleus. However, the exact mechanism by which nuclear PTEN is regulated remains unclear. We have recently reported that Plk1 specifically phosphorylates PTEN on Ser-380 during mitosis. Here we report that PTEN also localized to chromatin and that chromatin PTEN was removed by a proteasome-dependent process during mitotic exit. Pulldown analysis revealed that Cdh1, but not Cdc20, was significantly associated with PTEN. Cdh1 interacted with PTEN via two separate domains, and their interaction was enhanced by MG132, a proteasome inhibitor. Cdh1 negatively controlled the stability of chromatin PTEN by polyubiquitination. Phosphorylation of PTEN on Ser-380 impaired its interaction with Cdh1, thus positively regulating PTEN stability on chromatin. Significantly, the PTEN interaction with Cdh1 was phosphatase-independent, and Cdh1 knockdown via RNAi led to significant accumulation of chromatin PTEN, delaying mitotic exit. Combined, our studies identify Cdh1 as an important regulator of nuclear/chromatin PTEN during mitosis.     

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.     

An in vitro comparative assessment with a series of new triphenyltin(IV) 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates endowed with anticancer activities: structural modifications, analysis of efficacy and cytotoxicity involving human tumor cell lines

Four new triphenyltin(IV) complexes of composition Ph₃SnLH (where LH = 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoate) (1-4) were synthesized and characterized by spectroscopic (¹H, ¹³C and ¹¹⁹Sn NMR, IR, ¹¹⁹Sn Mössbauer) techniques in combination with elemental analysis. The ¹¹⁹Sn NMR spectroscopic data indicate a tetrahedral coordination geometry in non-coordinating solvents. The crystal structures of three complexes, Ph₃SnL¹H (1), Ph₃SnL³H (3), Ph₃SnL⁴H (4), were determined. All display an essentially tetrahedral geometry with angles ranging from 93.50(8) to 124.5(2)°; ¹¹⁹Sn Mössbauer spectral data support this assignment. The cytotoxicity studies were performed with complexes 1-4, along with a previously reported complex (5) in vitro across a panel of human tumor cell lines viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The screening results were compared with the results from other related triphenyltin(IV) complexes (6-7) and tributyltin(IV) complexes (8-11) having 2-/4-[(E)-2-(aryl)-1-diazenyl]benzoates framework. In general, the complexes exhibit stronger cytotoxic activity. The results obtained for 1-3 are also comparable to those of its o-analogs i.e. 4-7, except 5, but the advantage is the former set of complexes demonstrated two folds more cytotoxic activity for the cell line MCF-7 with ID₅₀ values in the range 41-53 ng/ml. Undoubtedly, the cytotoxic results of complexes 1-3 are far superior to CDDP, 5-FU and ETO, and related tributyltin(IV) complexes 8-11. The quantitative structure-activity relationship (QSAR) studies for the cytotoxicity of triphenyltin(IV) complexes 1-7 and tributyltin(IV) complexes 8-11 is also discussed against a panel of human tumor cell lines.     

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31–86%. Additional improvements were obtained by altering media formulations, resulting in bicistronic expression levels of 890, 1,800 and 1,010 nmol/L for 3A4, 2C9 and 1A2, respectively. The P450 titers achieved in fermentors exceeded those in flask fermentations by 3- to 6-fold in this study and up to 10-fold when compared with previously reported literature [FEBS Lett (1996) 397:210–214, Arch Biochem Biophys (1996) 327:254–259, Biochem Pharmacol (1998) 55:1315–1325, Drug Metab Pharmacokinet (2003) 18:42–47, Nat Biotechnol (1997) 15:784–788; Metab Eng (2000) 2:115–125]. Intact cells and isolated membranes obtained from 10-L fermentations were used to establish an efficient bioconversion system for the generation of metabolites. To demonstrate the utility of this approach, known metabolites of the anabolic steroid testosterone, the anti-inflammatory agent diclofenac and the analgesic agent phenacetin, were generated using 3A4, 2C9 and 1A2, respectively. The reaction conditions were optimized for pH, temperature, DO concentration, use of co-solvent and glucose supplementation. Conversion yields of 29–93% were obtained from 1-L reactions, enabling isolation of 59 mg 6-hydroxytestosterone, 110 mg 4-hydroxydiclofenac and 88 mg acetaminophen.     

Biochemical characterization of TRIM72 E3 ligase and its interaction with the insulin receptor substrate 1

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.     

AT2R − 1332 G:A polymorphism and its interaction with AT1R 1166 A:C,ACE I/D and MMP-9 − 1562 C:T polymorphisms: Risk factors for susceptibility to preeclampsia

The possible association of angiotensin type 2 receptor (AT2R) − 1332 G:A polymorphism with susceptibility to preeclampsia was studied in 252 women consisted of 155 women with preeclampsia and 97 healthy pregnant women. Also, the interaction of this polymorphism with angiotensin type 1 receptor (AT1R) 1166 A:C, angiotensin converting enzyme insertion/deletion (ACE I/D) and also with matrix metalloproteinase-9 (MMP-9) − 1562 C:T polymorphism was investigated. The AT2R − 1332 G:A polymorphism was detected using PCR–RFLP method. Significantly higher frequencies of GG+GA genotype and G allele of AT2R were observed in mild (80.2%, p = 0.003 and 47.5%, p = 0.012, respectively) and severe (77.8%, p = 0.034 and 48.1%, p = 0.026, respectively) preeclampsia compared to controls (60.8% and 35.1%, respectively). The presence of G allele was associated with 1.69-fold increased risk of preeclampsia (p = 0.005). In severe preeclamptic women, systolic and diastolic blood pressures in the presence of GG+GA genotype were significantly higher compared to those in the presence of AA genotype. The concomitant presence of both alleles of AT2R G and AT1R C was associated with 1.3 times increased risk of mild preeclampsia (p = 0.03). There was an interaction between AT2R G and ACE D alleles that significantly increased the risk of mild and severe preeclampsia by 1.38- and 1.3-fold, respectively. Also, interaction between MMP-9 T and AT2R G alleles increased the risk of severe preeclampsia 1.39-fold (p = 0.028). Our study demonstrated that the G allele of AT2R − 1332 G:A polymorphism is associated with an increased risk of preeclampsia. Also, epistatic interaction of G allele and each allele of the AT1R C, ACE D and MMP-9 T was associated with the risk of preeclampsia. Our findings suggest that the renin–angiotensin system (RAS) variants and gene–gene interactions affect the risk of preeclampsia.     

The MTMR9 rs2293855 polymorphism is associated with glucose tolerance,insulin secretion,insulin sensitivity and increased risk of prediabetes

Background

Polymorphism of rs2293855 in gene MTMR9 has been associated with obesity and metabolic syndrome. We aim to study the association of rs2293855 with type 2 diabetes mellitus (T2DM) intermediate phenotypes in a Han Chinese population.

Methods

The polymorphism was genotyped in 838 Han Chinese individuals using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS); all participants underwent a 75 g oral glucose tolerance test (OGTT); associations between the polymorphism and glucose tolerance, indices of insulin secretion and indices of insulin sensitivity were analyzed.

Results

The frequency of genotypes and alleles differed significantly between normal glucose tolerance and prediabetes (P = 0.043 and P = 0.009, respectively). The GG homozygous presented higher fasting plasma glucose (P = 0.009), higher 2-hour plasma glucose (P = 0.024) and higher glucose area under the curve (AUC, P = 0.01). Moreover, the G allele of rs2293855 was associated with glucose intolerance (fasting glucose, P = 0.012; glucose AUC, P = 0.006; 2-h glucose, P = 0.024); it is also associated with decreased indices of insulin sensitivity (fasting insulin, P = 0.043; insulin sensitivity index composite, P = 0.009; homeostasis model assessment of insulin resistance, HOMA-IR, P = 0.008) and decreased indices of insulin secretion (HOMA of beta cell function, HOMA-B, P = 0.028; insulinogenic index, P = 0.003). In addition, the minor allele G was also associated with increased risk of prediabetes (OR = 1.463, 95%CI: 1.066–2.009, P = 0.018).

Conclusions

Polymorphism of rs2293855 in MTMR9 is associated with measures of glucose tolerance, indices of insulin secretion and indices of insulin sensitivity. We also suggest that allele G is likely to increase the risk of prediabetes by influencing both insulin secretion and insulin sensitivity.     

De novo 3q22.1 q24 deletion associated with multiple congenital anomalies,growth retardation and intellectual disability

We describe a boy with a de novo deletion of 15.67 Mb spanning 3q22.1q24. He has bilateral micropthalmia, ptosis, cleft palate, global developmental delay and brain, skeletal and cardiac abnormalities. In addition, he has bilateral inguinal hernia and his right kidney is absent. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. This patient has some phenotypic features that are not shared by the other patients. More cases with smaller deletions defined by high resolution aCGH will enable better genotype–phenotype correlations and prioritizing of candidate genes for the identification of pathways and disease mechanisms.     

Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2

Leigh syndrome (LS) is a progressive neurodegenerative disease caused by either mitochondrial or nuclear DNA mutations resulting in dysfunctional mitochondrial energy metabolism. Mutations in genes encoding for subunits of the respiratory chain or assembly factors of respiratory chain complexes are often documented in LS cases. Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) enzyme deficiencies account for a significant proportion of mitochondrial disorders, including LS. In an attempt to expand the repertoire of known mutations accounting for LS, we describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in two complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for the R386C mutation in NDUFV1 and a frameshift mutation in the same gene, one patient was a compound heterozygote for the R88G and R199P mutations in NDUFV1, and two siblings were compound heterozygotes for an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity. The R386C, R88G, R199P, and E104A mutations were found to be likely pathogenic, and thus presumably account for the LS phenotype. This case series broadens our understanding of the etiology of LS by identifying new molecular defects that can result in complex I deficiency and may assist in targeted diagnostics and/or prenatal diagnosis of LS in the future.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.