Increased DNA breaks and up-regulation of both G1 and G2 checkpoint genes p21WAF1/CIP1 and 14-3-3 σ in circulating leukocytes of glaucoma patients and vasospastic individuals

Summary. Objective: Vascular disorder leading to local ischemia/reperfusion has been shown to play an important role in the glaucomatous damage. A decreased expression level of XPGC-gene has been found in circulating leukocytes of normal-tension glaucoma patients. Although decreased activity of XPGC-gene leads to insufficient DNA-repair, no leukopenia has been observed in glaucoma. Molecular mechanisms ensuring cell survival have not been elucidated yet for glaucoma with vascular disorder.Material and methods: Using the ex vivo optical imaging method of alkaline comet assay comparative quantification of DNA breaks was performed in circulating leukocytes of non-glaucomatous non-vasospastic and vasospastic individuals as well as both normal-tension and high-tension glaucoma patients. Relative expression levels of the anti-apoptotic factors P21^WAF1/CIP1 and 14-3-3 were investigated in all groups tested.Results and conclusions: The quantification of P21^WAF1/CIP1 showed the highest expression rates in high-tension glaucoma patients which were significantly higher than those in all other groups tested. The highest expression rates of 14-3-3 were found in both groups of glaucoma patients. These expression levels correlated well with DNA breaks measured. Since the expression of P21^WAF1/CIP1 in leukocytes was shown to be crucial for their survival under stress conditions, we suppose further that the up-regulation of this gene is the key event in the survival mechanisms of leukocytes in glaucoma accompanied with vascular disorder. The p21^WAF1/CIP1 gene should be further taken into consideration as a potential marker, the up-regulation of which in circulating leukocytes of vasospastic individuals may indicate an increased risk for the developing glaucoma.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Specificity and cooperativity at β-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions

Establishing a quantitative understanding of the determinants of affinity in protein–protein interactions remains challenging. For example, TEM‐1/β‐lactamase inhibitor protein (BLIP) and SHV‐1/BLIP are homologous β‐lactamase/β‐lactamase inhibitor protein complexes with disparate K_d values (3 nM and 2 μM, respectively), and a single substitution, D104E in SHV‐1, results in a 1000‐fold enhancement in binding affinity. In TEM‐1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV‐1 D104 does not in the wild type SHV‐1/BLIP co‐structure. Here, we present a 1.6 Å crystal structure of the SHV‐1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV‐1 D104 and BLIP K74 and a 400‐fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Synthesis of β-1, 3-Glucan and β-1, 3 and β-1, 4-Mixed Glucan from UDP-α-d-Glucose

A particulate enzyme preparation from Phaseolus aureus (mung bean) seedlings catalyzed the synthesis of a water insoluble β-1,3-glucan from UDP-α-d-glucose (UDPG) at high concentrations (0.4~20 mm) and an alkaline insoluble β-1,3 and β-1,4-mixed glucan from UDPG at a low concentration (8.5 µm).

Furthermore, the two kinds of β-glucan synthetases which were investigated with two reaction systems at high and low concentrations of UDPG had different properties in optimal pH, stability of enzyme activity, and metallic ion requirement.     

Interleukin-1β-31C/T and -511T/C Polymorphisms Were Associated with Preeclampsia in Chinese Han Population

Objective

The purpose of our study is to investigate the relationship between IL-1β -31C/T (rs1143627) and -511T/C (rs16944) polymorphisms and the preeclampsia (PE), and analyze the Linkage disequilibrium (LD) and haplotype frequency of the two polymorphism loci.

Methods

Polymorphisms at -31C/T and -511T/C of IL-1β were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) in 232 PE and 447 control subjects. Genotype and allele frequencies between case-control groups were compared by chi-square(X²) tests. Two-point LD and haplotype frequency analyses were done with the software Haploview4.2.

Results

Significant statistical differences were found between PE and control groups regarding genotype and allele frequencies of the two polymorphisms of IL-1β (For IL-1β -31C/T: X² = 11.478, P = 0.003; For IL-1β-511T/C: X² = 9.687, P = 0.008). LD analysis revealed that the IL-1β -31C/T SNP was in high LD with the IL-1β-511C/T SNP(D′ = 0.92, r² = 0.79). Both CT and TC haplotypes showed significant differences between case and control groups. Only the plasma level of Prothrombin Time had a significantly statistical difference among TT, CT and CC groups of the preeclamptic two polymorphisms of IL-1β-31C/T and -511T/C (for IL-1β-31C/T, F = 1.644, P = 0.01; F = 1.587, P = 0.016).

Conclusion

Our results revealed IL-1β was associated with the PE in Chinese Han population. The CT haplotype may increase the risk of PE, while haplotype TC could be considered as a protective haplotype of PE.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate     

Connexin-43 Interactions with ZO-1 and α- and β-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication, (ii) the ZO-1 ‘scaffold’ protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of α/β-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds α-tubulin equally well as β-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

Differential role of cyclooxygenase-1 and -2 on renal vasoconstriction to α1-adrenoceptor stimulation in normotensive and hypertensive rats

Aims

Hypertension is associated with the impairment of renal cyclooxygenase (COX) activity, which regulates vascular tone, salt and water balance and renin release. We aimed to evaluate the functional role of COX isoforms in kidneys isolated from spontaneously hypertensive rats (SHR) after α₁-adrenoceptor (α₁-AR) stimulation.

Main methods

Male six-month-old SHR and normotensive Wistar-Kyoto rats (WKY) were used. The kidneys were isolated to measure perfusion pressure and COX-1- or COX-2-derived prostanoids in response to α₁-AR activation.

Key findings

The basal perfusion pressure was higher in SHR kidneys compared with WKY kidneys (95 ± 11 vs. 68 ± 6 mm Hg, P < 0.05). Phenylephrine induced a greater vasopressor response in SHR kidneys (EC₅₀ of 1.89 ± 0.58 nmol) than WKY kidneys (EC₅₀ of 3.30 ± 0.54 nmol, P < 0.05 vs. SHR). COX-1 inhibition decreased the α₁-AR-induced vasoconstrictor response in WKY but did not affect SHR response, while COX-2 inhibition diminished the response in SHR. Both basal prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) values were higher in SHR kidney perfusates (P < 0.05) and were reduced by COX-1 and COX-2 inhibitors in both strains. Furthermore, phenylephrine increased PGI₂ through COX-2 in WKY and through COX-1 in SHR, but the agonist did not significantly modify TxA₂ in both strains.

Significance

The data suggest that COX-1contributes to vasoconstrictor effects in WKY kidneys and that COX-2 has the same effect in SHR kidneys. The results also suggest that basal release of COX-2-derived vasoconstrictor prostanoids is involved in renal vascular hypersensitivity in SHR.     

Synthesis and properties of alkylglucosides with mild detergent action: improved synthesis and purification of β-1-octyl-, -nonyl-, and -decyl-glucose. Synthesis of β-1-undecylglucose and β-1-dodecylmaltose

Medium chain β-1-alkylglycosides show interesting mild detergent properties. Therefore, their synthesis and purification have been investigated and improved so as to permit preparation of 50–100 g amounts. Preparatory methods are presented for the already known compounds β-1-octyl-, β-1-nonyl and β-1-decyl-glucose and for the new compounds β-1-undecylglucose and β-1-dodecylmaltose. Some relevant properties such as melting point, optical rotation, critical micelle concentration and NMR-spectra have been determined. They illustrate the suitability of this class of detergents for membrane research.     

Inhibition of the 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT)- and estradiol-mediated induction of rat uterine ornithine decarboxylase by prior treatment with o,p′DDT,estradiol, and tamoxifen

This study was designed to determine whether prior administration of inducers of rat uterine ornithine decarboxylase (ODC), such as 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT), estradiol-17β (E₂), or tamoxifen, inhibits the elevation of ODC by subsequently administered o,p′ DDT or estradiol-17β. o,p′ DDT (10 mg/day) was injected for 2 days to ovariectomized rats. One or two days later, when the levels of ODC returned to basal levels, o,p′ DDT (10 mg) and E₂ (0.05 μg) were administered intraperitoneally and, 6 or 5 h after these injections, uterine ODC was analyzed. The pretreatment with o,p′ DDT almost entirely blocked the induction of ODC by E₂ or o,p′ DDT. In another experiment, pretreatment with o,p′ DDT for 5 or 6 days eliminated the induction of ODC after injection of o,p′ DDT on Day 7. Similarly, the treatment of rats with the antiestrogen-tamoxifen (0.1 mg/day) for 4 days completely inhibited the subsequent elevation of ODC by either E₂ or o,p′ DDT administered on the fifth day. However, attempts to block the E₂-mediated elevation of ODC by prior treatment with E₂ yielded variable results. Two possibilities were considered to attempt to explain the mechanism of inhibition of induction of ODC by o,p′ DDT and tamoxifen: (a) induction of hepatic monooxygenase by these compounds, resulting in increased metabolism of the subsequently administered o,p′ DDT and E₂ into biologically less active components; (b) involvement of putrescine, the product of ODC action, in inhibiting ODC formation at the pretranslational level or at the post-translational level. It appears unlikely that the o,p′ DDT- and tamoxifen-mediated inhibition of ODC induction was due to an increase in hepatic biotransformation of o,p′ DDT and E₂. Pretreatment with tamoxifen or E₂ did not appear to induce the formation of hepatic microsomal cytochrome P-450, a component of the monooxygenase system. Furthermore, pretreatment with 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene (a compound with a structure similar to o,p′ DDT), which is not estrogenic but like o,p′ DDT elevates hepatic monooxygenase activity, did not inhibit E₂-or o,p′ DDT-mediated induction of uterine ODC. Concerning the possibility of putrescine inhibitory action, our observations that uterine diamine oxidase activity is negligible and that o,p′ DDT administration has no effect on this enzymatic activity suggested that elevation of ODC may result in higher levels of uterine putrescine or spermidine and spermine. The finding that the administration of putrescine to ovariectomized rats inhibited uterine ODC induction by o, p′ DDT supports the treatise that inhibition of ODC elevation after initial induction of ODC by antiestrogens and o,p′ DDT is due to putrescine- or polyamine-mediated inhibition of ODC. The possible mechanism of such product inhibition of ODC is disucssed.     

Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.     

EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.     

Synthesis of (±)-7,3′- and 7,4′-di-O-methyleriodictyol and of velutin and pilloin

Synthetic 2′-hydroxy-3,4′,6′-trimethoxy-4-benzyloxychalcone (I) affords (±)-7,3′-di-O-methyleriodictyol (II) and 7,3′-di-O-methylluteolin (or velutin, VII) identical with natural samples. Similarly synthetic 2′-hydroxy-4,4′,6′-trimethoxy-3-benzyloxychalcone (X) gives natural (±)-7,4′-di-O-methyleriodictyol (XI) and 7,4′-di-O-methylluteolin (or pilloin, IX). However, attempts to partially etherify II with one mole of prenyl bromide to obtain the natural prenyl ether failed; only the corresponding diprenyloxychalcone (IV) was obtained.     

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional β-D-xylosidase/α-L-arabinofuranosidase

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-β-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional β-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.     

Synthesis and biological evaluation of 3-(azolylmethyl)-1H-indoles and 3-(α-azolylbenzyl)-1H-indoles as selective aromatase inhibitors

This present study identifies a number of azolyl-substituted indoles as potent inhibitors of aromatase. In the sub-series of 3-(azolylmethyl)-1H-indoles, four imidazole derivatives and their triazole analogues were tested. Imidazole derivatives 11 and 14 in which the benzyl moiety was substituted by 2-chloro and 4-cyano groups, respectively, were the most active, with IC₅₀ values ranging between 0.054 and 0.050 μM. In the other sub-series, eight 3-(α-azolylbenzyl)-1H-indoles were prepared and tested. Compound 30, the N-ethyl imidazole derivative, proved to be an aromatase inhibitor, showing an IC₅₀ value of 0.052 μM. All target compounds were further evaluated against 17α-hydroxylase/C17,20-lyase to determine their selectivity profile.