Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

A quantitative coronary angiography-matched comparison between a prospective randomised multicentre cutting balloon angioplasty and bare metal stent trial (REDUCE III) and the Rapamycin-Eluting Stent Evaluation At Rotterdam Cardiology Hospital (RESEARCH) study

Aims: There remains significant concern about the long-term safety of drug-eluting stents (DES). However, bare metal stents (BMS) have been used safely for over two decades. There is therefore a pressing need to explore alternative strategies for reducing restenosis with BMS. This study was designed to examine whether IVUS-guided cutting balloon angioplasty (CBA) with BMS could convey similar restenosis rates to DES. Methods and results: In the randomised REstenosis reDUction by Cutting balloon angioplasty Evaluation (REDUCE III) study, 521 patients were divided into four groups based on device and IVUS use before BMS (IVUS-CBA-BMS: 137 patients; Angio-CBA-BMS: 123; IVUS-BA-BMS: 142; and Angio-BA-BMS: 119). At follow-up, the IVUS-CBA-BMS group had a significantly lower restenosis rate (6.6%) than the other groups (p=0.016). We performed a quantitative coronary angiography (QCA) based matched comparison between an IVUS-guided CBA-BMS strategy (REDUCE III) and a DES strategy (Rapamycin-Eluting-Stent Evaluation At Rotterdam Cardiology. Hospital, the RESEARCH study). We matched the presence of diabetes, vessel size, and lesion severity by QCA. Restenosis (>50% diameter stenosis at follow-up) and target vessel revascularisation (TVR) were examined. QCA-matched comparison resulted in 120-paired lesions. While acute gain was significantly greater in IVUS-CBA-BMS than DES (1.65 +/- 0.41 mm vs. 1.28 +/- 0.57 mm, p=0.001), late loss was significantly less with DES than with IVUS-CBA-BMS (0.03 +/- 0.42 mm vs. 0.80 +/- 0.47 mm, p=0.001). However, no difference was found in restenosis rates (IVUS-CBA-BMS: 6.6% vs. DES: 5.0%, p=0.582) and TVR (6.6% and 6.6%, respectively). Conclusions: An IVUS-guided CBA-BMS strategy yielded restenosis rates similar to those achieved by DES and provided an effective alternative to the use of DES.

Protein crystallography and examples of its applications in medicinal chemistry

The field of protein crystallography inspires and enthrals, whether it be for the beauty and symmetry of a perfectly formed protein crystal, the unlocked secrets of a novel protein fold, or the precise atomic-level detail yielded from a protein-ligand complex. Since 1958, when the first protein structure was solved, there have been tremendous advances in all aspects of protein crystallography, from protein preparation and crystallisation through to diffraction data measurement and structure refinement. These advances have significantly reduced the time required to solve protein crystal structures, while at the same time substantially improving the quality and resolution of the resulting structures. Moreover, the technological developments have induced researchers to tackle ever more complex systems, including ribosomes and intact membrane-bound proteins, with a reasonable expectation of success. In this review, the steps involved in determining a protein crystal structure are described and the impact of recent methodological advances identified. Protein crystal structures have proved to be extraordinarily useful in medicinal chemistry research, particularly with respect to inhibitor design. The precise interaction between a drug and its receptor can be visualised at the molecular level using protein crystal structures, and this information then used to improve the complementarity and thus increase the potency and selectivity of an inhibitor. The use of protein crystal structures in receptor-based drug design is highlighted by (i) HIV protease, (ii) influenza virus neuraminidase and (iii) prostaglandin H-2-synthetase. These represent, respectively, examples of protein crystal structures that (i) influenced the design of drugs currently approved for use in the treatment of HIV infection, (ii) led to the design of compounds currently in clinical trials for the treatment of influenza infection and (iii) could enable the design of highly specific non-steroidal anti-inflammatory drugs that lack the common side-effects of this drug class.

Pentoxifylline modifies three-dimensional collagen lattice model contraction and expression of collagen types I and III by human fibroblasts derived from post-burn hypertrophic scars and from normal skin

Fibroblasts are thought to be partially responsible for the persisting contractile forces that result in burn contractures. Using a monolayer cell culture and fibroblast populated collagen lattice (FPCL) three-dimensional model we subjected hypertrophic scar and non-cicatricial fibroblasts to the antifibrogenic agent pentoxifylline (PTF - 1 mg/mL) in order to reduce proliferation, collagen types I and III synthesis and model contraction. Fibroblasts were isolated from post-burn hypertrophic scars (HSHF) and non-scarred skin (NHF). Cells were grown in monolayers or incorporated into FPCL`s and exposed to PTF. In monolayer, cell number proliferation was reduced (46.35% in HSHF group and 37.73% in NHF group, p < 0.0001). PTF selectively inhibited collagen III synthesis in the HSHF group while inhibition was more evident to type I collagen synthesis in the NHF group. PTF also reduced contraction in both (HSHF and NHF) FPCL. (C) 2009 Elsevier Ltd and ISBI. All rights reserved.

Gold(III) template synthesis of a pendant-arm macrocycle

Gold(III)-directed condensation of ethane-1,2-diamine with nitroethane and formaldehyde yielded the gold-coloured macrocyclic complex (cis-6,13-dimethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecan-1-ido)gold(III) and the orange acyclic complex (1,9-diamino-5-methyl-5-nitro-3,7-diazanoran-3-ido)gold(III) in good yields. Dissolution in strongly acidic solution gave the colourless fully protonated complexes. The pendant nitro groups are disposed on the same side of the macrocycle in a cis geometry, as confirmed by crystal structure analysis. In both complexes the gold ion lies in a square-planar environment of four nitrogen donors, and the co-ordinate bond to the deprotonated amine is shorter than the remaining Au-N distances.

Acute Pancreatitis Hypertonic Saline Increases Heat Shock Proteins 70 and 90 and Reduces Neutrophil Infiltration in Lung Injury

Objectives: Acute pancreatitis (AP) protease release induces lung parenchymal destruction via matrix metalloproteinases (MMPs), a neutrophil (polymorphonuclear leukocyte)-dependent process. Recent studies in hemorrhagic shock revealed that hypertonic saline (HTS) has an anti-inflammatory effect and can inhibit a variety of neutrophil functions. The aim of this study was to determine whether HTS and its actions in the pathway of neutrophil migration, MMPs, and heat shock proteins (HSPs) are effective in protecting the lung from injury associated with AP. Methods: We determined neutrophil infiltration and expressions of MMPs and HSPs in the lung tissue after AP induced by retrograde infusion of 2.5% of sodium taurocholate. Results: Animals submitted to AP that received HTS compared with those who received normal saline presented with increased HSP70 and HSP90 expressions and reduced myeloperoxidase levels and MMP-9 expression and activity. Conclusions: Our data raised the hypothesis that a sequence of HTS lung protection events increases HSP70 and HSP90, inhibiting infiltration of neutrophils and their protease actions in the lung.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis

P>We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.

Structural and electron self-exchange rate variations in isomeric (hexaamine)cobalt(III/II) complexes

The syntheses and characterisation of the new macrocyclic hexaamine trans-(5(S),7(S),12(R),14(R)-tetramethyl)-1,4,8,11-tetraazacyclotetradecane-6,13-diamine (L-6) and its Co-III complex are reported. The X-ray crystal structural analyses of [CoL6]Cl-2(ClO4) [monoclinic, space group C2/c, a = 16.468(3) Angstrom, b = 9.7156(7) Angstrom, c = 15.070(3) Angstrom, beta = 119.431(8)degrees, Z = 4] and the closely related cis-diamino-substituted macrocyclic complex [CoL2](ClO4)(3) . 2H(2)O (L-2 = cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine) [orthorhombic, space group Pna2(1), a = 16.8220(8) Angstrom, b = 10.416(2) Angstrom, c = 14.219(3) Angstrom, Z = 4] reveal significant variations in the observed Co-N bond lengths and coordination geometries, which may be attributed to the trans or cis disposition of the pendent primary amines. The Co-III/II self-exchange electron transfer rate constants for these and other closely related hexaamines have been determined, and variations of some 2 orders of magnitude are found between pairs of trans and cis isomeric Co-III complexes.

Five-Year Follow-Up of a Randomized Comparison Between Off-Pump and On-Pump Stable Multivessel Coronary Artery Bypass Grafting. The MASS III Trial

Background-Coronary artery bypass graft surgery with cardiopulmonary bypass is a safe, routine procedure. Nevertheless, significant morbidity remains, mostly because of the body`s response to the nonphysiological nature of cardiopulmonary bypass. Few data are available on the effects of off-pump coronary artery bypass graft surgery (OPCAB) on cardiac events and long-term clinical outcomes. Methods and Results-In a single-center randomized trial, 308 patients undergoing coronary artery bypass graft surgery were randomly assigned: 155 to OPCAB and 153 to on-pump CAB (ONCAB). Primary composite end points were death, myocardial infarction, further revascularization (surgery or angioplasty), or stroke. After 5-year follow-up, the primary composite end point was not different between groups (hazard ratio 0.71, 95% CI 0.41 to 1.22; P=0.21). A statistical difference was found between OPCAB and ONCAB groups in the duration of surgery (240 +/- 65 versus 300 +/- 87.5 minutes; P<0.001), in the length of ICU stay (19.5 +/- 17.8 versus 43 +/- 17.0 hours; P<0.001), time to extubation (4.6 +/- 6.8 versus 9.3 +/- 5.7 hours; P<0.001), hospital stay (6 +/- 2 versus 9 +/- 2 days; P<0.001), higher incidence of atrial fibrillation (35 versus 4% of patients; P<0.001), and blood requirements (31 versus 61% of patients; P<0.001), respectively. The number of grafts per patient was higher in the ONCAB than the OPCAB group (2.97 versus 2.49 grafts/patient; P<0.001). Conclusions-No difference was found between groups in the primary composite end point at 5-years follow-up. Although OPCAB surgery was related to a lower number of grafts and higher episodes of atrial fibrillation, it had no significant implications related to long-term outcomes.

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we describe the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution. (C) 1997 Elsevier Science B.V.

A randomized comparative study of patients undergoing myocardial revascularization with or without cardiopulmonary bypass surgery: The MASS III Trial

The MASS III Trial is a large project from a single institution, The Heart Institute of the University of Sao Paulo, Brazil (InCor), enrolling patients with coronary artery disease and preserved ventricular function. The aim of the MASS III Trial is to compare medical effectiveness, cerebral injury, quality of life, and the cost-effectiveness of coronary surgery with and without of cardiopulmonary bypass in patients with multivessel coronary disease referred for both strategies. The primary endpoint should be a composite of cardiovascular mortality, cerebrovascular accident, nonfatal myocardial infarction, and refractory angina requiring revascularization. The secondary end points in this trial include noncardiac mortality, presence and severity of angina, quality of life based on the SF-36 Questionnaire, and cost-effectiveness at discharge and at 5-year follow-up. In this scenario, we will analyze the cost of the initial procedure, hospital length of stay, resource utilization, repeat hospitalization, and repeat revascularization events during the follow-up. Exercise capacity will be assessed at 6-months, 12-months, and the end of follow-up. A neurocognitive evaluation will be assessed in a subset of subjects using the Brain Resource Center computerized neurocognitive battery. Furthermore, magnetic resonance imaging will be made to detect any cerebral injury before and after procedures in patients who undergo coronary artery surgery with and without cardiopulmonary bypass.

Sialylation of beta 1 Integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta 1 integrins from human colon tumors have elevated levels of alpha 2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta 1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha 2-6 sialylation of the beta 1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha 2-6 sialylation exhibit beta 1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha 2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha 2-6-sialylated, beta 1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes ( unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta 1 subunit, suggesting that beta 1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.