The effect of cigarette smoking on radiographic bone healing after elective foot surgery.

UNLABELLED: This study aims to compare radiographic healing rates of Austin bunionectomies in smokers, nonsmokers, and secondhand smokers. Delayed bone healing has been linked to cigarette smoking previously, but no study is known to have examined smoking in relation to elective foot surgery. We hypothesized that smoking will delay bone healing after elective foot surgery. Surgical patients were placed into 1 of 3 cigarette smoking-related groups. Nicotine dependence was measured by the standardized modified Fagerström est and a urine cotinine test. Bone healing was determined via examination of postsurgical radiographs. Outcomes were assessed with 1-way analyses of variance. Forty-six patients were prospectively evaluated. There were 17 smokers, 12 secondhand smokers, and 17 nonsmokers. Healing time after Austin bunionectomy was 69 days (SD = 26.0), 120 days (SD = 55.3), and 78 days (SD = 19.1) in nonsmokers, smokers, and secondhand smokers, respectively. It was noted that as urine cotinine number increased, the healing time also increased (Pearson correlation = -.314, P < .01). The same was noted with the score associated with the Fagerström questionnaire, showing an increase in healing time with an increase in score (Pearson correlation = -.128, P < .05). The osteotomy of a smoker took 1.73 times longer to reach radiographic bone consolidation than that of a nonsmoker. This equates to a 42% increase in time to bone healing in the smoking patient. Increased healing time was also correlated to increased urine cotinine and a higher Fagerström number. Smoking is shown to delay radiographic healing. LEVEL OF CLINICAL EVIDENCE: 2.

Extraction and Isolation of Antifreeze Proteins from Winter Rye (Secale cereale L.) Leaves.

Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing.

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.

Effects of adenosine receptor agonists on nitric oxide release in mouse during endotoxemia.

The effects of adenosine receptor agonists on plasma NOx- (NO2- and NO3-) production in mice treated with lipopolysaccharide (LPS) were investigated. NOx- is the stable decomposition product of nitric oxide (NO), which can be measured as a marker of NO production. Injection of the mice with LPS resulted in increased plasma NOx- concentration, reaching a peak after 8 hr (38 times basal level) and then declining slowly. Pretreatment with the adenosine agonists R-phenylisopropyladenosine (R-PIA), 5'-N-ethylcarboxamidoadenosine (NECA), 5'-(N-cyclopropyl)carboxamidoadenosine (CPCA) and N6-cyclohexyladenosine (CHA) 1 hr before LPS administration caused a dose-dependent reduction of plasma NOx- concentration. The rank order of inhibitory potency was NECA > or = R-PIA > CPCA > CHA, which is characteristic of neither A1 nor A2 receptors.

Pre- and posttraining infusion of N-methyl-D-aspartate receptor antagonists into the amygdala impair memory in an inhibitory avoidance task.

Involvement of amygdaloid N-methyl-D-aspartate (NMDA) receptors in memory processes was investigated. Rats with cannulas implanted in the basolateral amygdala were trained on a 1 trial step-through inhibitory avoidance task and tested for 24-hr retention. Pretraining infusion of 2-amino-5-phosphonovaleric acid (APV) into the amygdala, but not striatum or hippocampus, produced a dose-dependent retention deficit, which was attenuated by immediate posttraining intra-amygdala infusion of NMDA. Posttraining APV infusion also caused a dose- and time-dependent retention deficit. Pretest APV infusion had no effect on performance in the retention test. Further, pre- or posttraining infusion of 5.0 micrograms APV failed to affect acquisition and retention in the Morris water maze task. These findings suggest that amygdala NMDA receptors are normally activated by aversive training and play a critical role in memory formation for affective experience.

Expression of inducible nitric oxide synthase in mice: pharmacological evaluation of adenosine receptor agonists.

Inhibition of inducible nitric oxide (NO) synthase during endotoxaemia may be of therapeutic value. We have previously shown that pretreatment of mice with adenosine receptor agonists 1 h before lipopolysaccharide administration results in a dose-dependent reduction of plasma nitrite and nitrate (NOx-) levels. This report examines the effects of adenosine receptor agonists, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclohexyladenosine (CHA), R-phenylisopropyl-adenosine (R-PIA) and 5'-(N-cyclopropyl)carboxamidoadenosine (CPCA), on the level of inducible NO synthase expression in a model of liver inflammation induced by lipopolysaccharide. Following lipopolysaccharide administration (10 mg/kg, i.p.), liver mRNA expression peaked at 3 h and declined to 35% of maximal level after 24 h. Pretreatment with adenosine receptor agonists (0.001 mg/kg to 5 mg/kg, i.p.) depressed inducible NO synthase mRNA expression significantly. Down-regulation of inducible NO synthase mRNA expression corresponded with changes in plasma NOx- level as well as activity of NO synthase in the liver. Administration of R-PIA (5 mg/kg, i.p.) increased the survival of animals injected with a lethal dose of lipopolysaccharide. Thus adenosine receptor agonists may useful as anti-inflammatory agents in the treatment of endotoxaemia.

Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation.

Stonustoxin (SNTX) is a 148,000 mol. wt lethal factor isolated from Synanceja horrida venom. It induces species-restricted red cell haemolysis which correlates with its effects on platelet aggregation in whole blood. SNTX induced a concentration-dependent and irreversible platelet aggregation in rabbit or rat but not in human or mouse whole blood. The degree of haemolysis and platelet aggregation induced by SNTX in rabbit or rat whole blood were concentration dependent. At concentrations of SNTX where only slight or no haemolysis was observed, no platelet aggregation occurred. Although SNTX itself could not induce platelet aggregation in rabbit or rat platelet-rich plasma (PRP), it had biphasic effects on collagen- or ADP-induced platelet aggregation in PRP. It inhibited collagen- or ADP-induced platelet aggregation at high concentrations (0.08-0.8 micrograms/ml) but the response was potentiated by lower stonustoxin concentrations (0.008-0.016 micrograms/ml). The inhibition of collagen- or ADP-induced platelet aggregation might be due to the lytic activity of SNTX on the platelets.

Adenosine and its receptor agonists potentiate nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages.

The effect of adenosine and its agonists on nitric oxide synthase (NOS) activity and the production of nitrite induced by lipopolysaccharide (LPS) in RAW 264.7 cells were investigated. Nitrite production and NOS activity in the RAW 264.7 cells were increased up to 2.5 fold after co-exposure of the cells to LPS and adenosine or its agonists, as compared to LPS alone. Adenosine and its agonists had no effect on NOS activity when incubated alone with RAW 264.7 cells. Enhancement caused by adenosine or its agonists was dose-dependent but the effect was neither A1 nor A2 receptor specific. These findings suggest that during pathological conditions such as inflammation or trauma, the significant amounts of cellular adenosine which are released may increase the production of NO by macrophages.

Effects of alpha-momorcharin, beta-momorcharin and alpha-trichosanthin on lipogenesis and testicular and adrenal steroidogenesis in vitro and plasma-glucose levels in vivo.

The effects of alpha-momorcharin, beta-momorcharin and alpha-trichosanthin on lipogenesis in isolated rat adipocytes were examined. None of the three abortifacient proteins possessed lipogenic activity. The plant proteins did not affect the plasma-glucose level in fasting mice nor did they affect luteinizing hormone-induced testosterone production in isolated rat Leydig cells or corticotropin-induced corticosterone production in isolated rat adrenal decapsular cells by the end of a 2-h incubation period. The results suggest that the functions of adipocytes, adrenal decapsular cells, Leydig cells and pancreatic beta cells were not greatly affected after short-term exposure to the abortifacient proteins.

Histochemical method for detecting nitric oxide synthase activity in cell cultures.

NADPH diaphorase histochemistry has been used extensively for detecting nitric oxide synthase (NOS) activity in various cell types including neuronal cell bodies, vascular endothelium, cells of the immune system and epithelial cells. The use of the diaphorase technique in cell cultures to study the induction of NOS has not been investigated. In this paper we report the use of diaphorase histochemistry as a good marker for the detection of NOS activity in cultured cells. This technique can be used in conjunction with other established techniques to determine the presence and activity of NOS in cultured cells.

Progesterone facilitates thrust in female hamsters implanted with estradiol at the medial preoptic area.

Progesterone (P) facilitates male-like sexual behavior in female hamsters primed with estrogen. Since estrogen plus P activates lordosis in female hamsters and the site of the hormonal action is at the ventromedial hypothalamus (VMH), it is possible that the same hormones act at the same site to induce male-like sexual behavior. However, VMH lesion abolishes lordosis, but not thrust, in ovariectomized female hamsters treated with estrogen plus P. Therefore, we tried another brain site--the medial preoptic area (mPOA). We implanted cannula, 27 gauge and filled with estradiol at the tip, at the mPOA or the VMH bilaterally in ovariectomized female hamsters. The hamsters received subcutaneous injections of P (0.5 mg in 0.05 ml sesame oil) at noon every fourth day. Behavior tests were conducted the same night of the injection day and the day before. The procedure was repeated five times. During a test night, each experimental female hamster was paired with a male hamster first and then with another estrous female. Recorded behavioral events were duration of lordosis and frequency of thrust. Both mPOA and VMH groups displayed lordosis only on the day of P injection, without any significant group difference. As to the male-like sexual behavior, 9 out of 54 mPOA females displayed thrust at least once but none of 27 VMH females ever did. Furthermore, 7 of the 9 females displayed thrust only on the day of the P injection. We conclude that the mPOA was also the site for the activation of male sex behavior in female hamsters. However, the hormones acting at the mPOA to induce thrusts were estrogen plus P.

Involvement of hippocampal NMDA and AMPA receptors in acquisition, formation and retrieval of spatial memory in the Morris water maze.

This study investigated the roles of hippocampal N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptors in acquisition, consolidation and retrieval processes of spatial memory. Male Wistar rats with indwelling cannulae in the dorsal hippocampus received 4 training trials on the Morris water maze for consecutively 6 days. Rats received infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) or the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) into the hippocampus under one of the three schedules: 5 min prior to each daily training session, immediately after each daily training session or 5 min prior to the final testing trial. Pretraining intra-hippocampal infusion of 5.0 micrograms AP5 retarded acquisition. The same dose of AP5 given after training had little effect but a higher dose (10.0 micrograms) did slow down progress in the acquisition curve. Pretest infusion AP5 failed to affect memory retrieval. Pretraining intra-hippocampal infusion of 1.0 micrograms CNQX also impaired acquisition, but posttraining infusion of CNQX at 1.0 or 2.0 micrograms had no effect. However, pretest infusion of 1.0 micrograms CNQX markedly impaired retrieval of the already-formed spatial memory. These findings taken together suggest that acquisition in a spatial task involves hippocampal NMDA and AMPA receptors, consolidation of spatial memory involves NMDA receptors and retrieving such memory involves AMPA receptors.

Nitric oxide in septic shock: directions for future therapy?

Septic shock is a major cause of death among patients in intensive care units. It has a mortality rate of 20% to 80%. The clinical syndrome of septic shock is characterised by hypotension, hyporesponsiveness to vasoconstrictors and volume depletion which will then lead to multiorgan dysfunction and death. Except for surgical and supportive care, no specific therapy is known. Recently interest has been focused on the role of nitric oxide (NO) in septic shock. Large amounts of NO released by the endothelium and vascular smooth muscle cells lead to profound vasodilation and hyporesponsiveness to vasoconstrictors. The cytotoxic effect of NO could also cause tissue injury and organ failure. Inhibition of NO synthase, the enzyme responsible for NO production, has been proposed as a new therapy for septic shock. However, experimental reports have provided conflicting results, demonstrating both beneficial and detrimental effects. A brief review of the role of NO in septic shock and the possible use of NO synthase inhibitors as potential therapeutic agents is presented here.

Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases.

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases, occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid-binding activities for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110α/p85α. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85α N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110α. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85α-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110α. Phosphopeptide binding to p110ß/p85α and p110δ/p85α complexes also induces their lipid binding. We present a crystal structure of WT p110α complexed with the p85α inter-SH2 domain and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110α consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110α and p85α mutations with signalling potential.

Association and interaction effect between VEGF receptor-2 (VEGFR-2) gene polymorphisms and dietary pattern on blood uric acid in Malays and Indians.

INTRODUCTION: Gout and hyperuricaemia attributed to genetic and lifestyle factors have been associated with several chronic diseases. This study aimed to determine the association and interaction effects between vascular endothelial growth factor receptor-2 (VEGFR-2) gene polymorphisms (rs1870377 and rs2071559) and dietary patterns on blood uric acid in Malay and Indian adults. METHODS: Dietary intakes of 153 Malays and 177 Indians were obtained using a food frequency questionnaire for the construction of dietary patterns using factor analysis. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Anthropometric measurements, body mass index (BMI) and blood pressure and biomarkers, uric acid, glycated haemoglobin A1c (HbA1c), and blood lipids were determined. RESULTS: There were significant differences in the mean values for HbA1c (41 +/- 12 vs 45 +/- 8 mmol/mol, p < 0.001) and blood lipids levels (p < 0.05) between Malays and Indians. Significant correlations were obtained between uric acid with selected blood lipids (p < 0.05) and BMI in Malays (r = 0.362, p < 0.001) and Indians (r = 0.212, p < 0.01). Four dietary patterns were extracted from dietary intakes of all subjects: 'Vegetables diet'; 'Fruits diet' (FD); 'Animal protein and rice diet'; and 'Fast foods and preserved foods diet'. There were no significant associations between dietary patterns (p = 0.054-0.609) and VEGFR-2 gene polymorphisms (p = 0.348-0.778) with uric acid. In Malay subjects, the interaction of rs2071559 and FD had a borderline effect (p = 0.05) on blood uric acid after adjusting for potential confounders. CONCLUSION: The associations and gene-diet interactions involving VEGFR-2 gene polymorphisms and FD on uric acid provide new information on gout and hyperuricaemia risks in Malays.

An efficient alignment algorithm for searching simple pseudoknots over long genomic sequence.

Structural alignment has been shown to be an effective computational method to identify structural noncoding RNA(ncRNA) candidates as ncRNAs are known to be conserved in secondary structures. However, the complexity of the structural alignment algorithms becomes higher when the structure has pseudoknots. Even for the simplest type of pseudoknots (simple pseudoknots), the fastest algorithm runs in O(mn3) time, where m, n are the length of the query ncRNA (with known structure) and the length of the target sequence (with unknown structure), respectively. In practice, we are usually given a long DNA sequence and we try to locate regions in the sequence for possible candidates of a particular ncRNA. Thus, we need to run the structural alignment algorithm on every possible region in the long sequence. For example, finding candidates for a known ncRNA of length 100 on a sequence of length 50,000, it takes more than one day. In this paper, we provide an efficient algorithm to solve the problem for simple pseudoknots and it is shown to be 10 times faster. The speedup stems from an effective pruning strategy consisting of the computation of a lower bound score for the optimal alignment and an estimation of the maximum score that a candidate can achieve to decide whether to prune the current candidate or not.

Opiate-like and adrenocorticotrophin-like materials in equine pancreas.

Equine pancreatic acetone powder was extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to salt fractionation, gel filtration and chromatography on CM-cellulose. Steroidogenic activity, ACTH-like immunoreactivity and opiate receptor binding activity were distributed among the CM-cellulose chromatographic fractions derived from material unretarded as well as from material retarded on Sephadex G-25. The data indicates a separation of steroidogenic and opiate receptor binding activities, and the presence of opiate-like molecules with different affinities of binding to mu and delta opiate receptors.

Hormones with adrenocorticotropic and opiate-like activities from the carp (Cyprinus carpio) pituitary.

Carp (Cyprinus carpio) pituitary acetone powder was extracted with a mixture of water, hydrochloric acid and acetone. An acid acetone powder was formed by adding the pituitary extract into a large volume of chilled acetone and subsequently recovering the precipitate. The powder was subjected to ion exchange chromatography on CM cellulose. Fractions adsorbed on the ion exchanger exhibited ACTH-like activity as evidenced in the ability to stimulate lipolysis in isolated hamster adipocytes and corticosterone production in isolated rat adrenal decapsular cells and also in cross-reactivity in an ACTH-specific radioimmunoassay. A portion of the ACTH-like bioactivity and immunoactivity was unadsorbed on the ion exchanger. Opiate-like activity in opiate receptor binding assay, employing [3H]D-ala2-D-leu5 enkephalin or [3H]naloxone as ligand, also resided in fractions adsorbed on CM cellulose. The data indicate a separation of ACTH-like and opiate-like activities, and the presence of opiate-like molecules with different affinities of binding to mu and delta opiate receptors.

Presence of corticotropin-like and opiate-like hormones in rat and bovine placental tissues.

Acid acetone powder of rat placentas was fractionated on Sephadex G-25 into a void volume peak (R-1) and three retarded peaks (R-2, R-3 and R-4). R-3 contained opiate-like activity and R-4 corticotropin-like activity, suggesting that separate corticotropin-like and opiate-like activities with molecular weight smaller than 5000 were present in rat placentas. Acid acetone powder of bovine placentas contained opiate-like activity which was unretarded on Sephadex G-25. Acid acetone powder of rat brains but not those of lungs, livers or kidneys possessed opiate receptor binding and steroidogenic activities, indicating that the activities in placentas were not due to enzymatically generated artifacts or to peptides contained in blood trapped in the organs.