Coronary artery hypoxic vasorelaxation is augmented by perivascular adipose tissue through a mechanism involving hydrogen sulphide and cystathionine-ß-synthase.

AIM: Hypoxia causes vasodilatation of coronary arteries which protects the heart from ischaemic damage through mechanisms including the generation of hydrogen sulphide (H2 S), but the influence of the perivascular adipose tissue (PVAT) and myocardium is incompletely understood. This study aimed to determine whether PVAT and the myocardium modulate the coronary artery hypoxic response and whether this involves hydrogen sulphide. METHODS: Porcine left circumflex coronary arteries were prepared as cleaned segments and with PVAT intact, myocardium intact or both PVAT and myocardium intact, and contractility investigated using isometric tension recording. Immunoblotting was used to measure levels of H2 S-synthesizing enzymes: cystathionine-ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (MPST). RESULTS: All three H2 S-synthesizing enzymes were detected in the artery and myocardium, but only CBS and MPST were detected in PVAT. Hypoxia elicited a biphasic response in cleaned artery segments consisting of transient contraction followed by prolonged relaxation. In arteries with PVAT intact, hypoxic contraction was attenuated and relaxation augmented. In arteries with myocardium intact, hypoxic contraction was attenuated, but relaxation was unaffected. In replacement experiments, replacement of dissected PVAT and myocardium attenuated artery contraction and augmented relaxation to hypoxia, mimicking the effect of in situ PVAT and indicating involvement of a diffusible factor(s). In arteries with intact PVAT, augmentation of hypoxic relaxation was reversed by amino-oxyacetate (CBS inhibitor), but not DL-propargylglycine (CSE inhibitor) or aspartate (inhibits MPST pathway). CONCLUSION: PVAT augments hypoxic relaxation of coronary arteries through a mechanism involving H2 S and CBS, pointing to an important role in regulation of coronary blood flow during hypoxia.

A critical role for cystathionine-ß-synthase in hydrogen sulfide-mediated hypoxic relaxation of the coronary artery.

Hypoxia-induced coronary artery vasodilatation protects the heart by increasing blood flow under ischemic conditions, however its mechanism is not fully elucidated. Hydrogen sulfide (H2S) is reported to be an oxygen sensor/transducer in the vasculature. The present study aimed to identify and characterise the role of H2S in the hypoxic response of the coronary artery, and to define the H2S synthetic enzymes involved. Immunoblotting and immunohistochemistry showed expression of all three H2S-producing enzymes, cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), in porcine coronary artery. Artery segments were mounted for isometric tension recording; hypoxia caused a transient endothelium-dependent contraction followed by prolonged endothelium-independent relaxation. The CBS inhibitor amino-oxyacetate (AOAA) reduced both phases of the hypoxic response. The CSE inhibitor dl-propargylglycine (PPG) and aspartate (limits MPST) had no effect alone, but when applied together with AOAA the hypoxic relaxation response was further reduced. Exogenous H2S (Na2S and NaHS) produced concentration-dependent contraction followed by prolonged relaxation. Responses to both hypoxia and exogenous H2S were dependent on the endothelium, NO, cGMP, K+ channels and Cl-/HCO3- exchange. H2S production in coronary arteries was blocked by CBS inhibition (AOAA), but not by CSE inhibition (PPG). These data show that H2S is an endogenous mediator of the hypoxic response in coronary arteries. Of the three H2S-producing enzymes, CBS, expressed in the vascular smooth muscle, appears to be the most important for H2S generated during hypoxic relaxation of the coronary artery. A contribution from other H2S-producing enzymes only becomes apparent when CBS activity is inhibited.

Filariasis in axillary lymph node.

A case of adult filarial worms detected in an axillary lymph node of an asymptomatic patient. A 64 year-old Indian female underwent a mammogram and was incidentally found to have punctate microcalcifications in the upper outer quadrant of the left breast with left axillary lymphadenopathy. She has underlying hypertension and diabetes mellitus on oral medications. She has no family history of breast malignancy. Fine needle aspiration of the left axillary lymph node was suggestive of reactive lympha-denitis. Histopathological examination of excisional biopsy of left breast lump showed fibrocystic disease; no evidence of malignancy was detected whereas excisional biopsy of left axillary lymph node showed reactive lymphoid hyperplasia, featuring variably sized lymphoid follicles with intact mantle zone. No expansion of marginal zone was noted. Occasional pigment-laden macrophages were seen. One of the lymph node showed presence of calcified serpinginous tubular bodies, in keeping with non viable parasite organisms with intact outlines of the structures. There were no eosinophilic infiltrates. The possibility of filarial infestation was suspected. Histopathological sample was sent for further identification and confirmed the presence of adult filarial worm.

Suppression of peptide ion dissociation under electron capture: role of backbone amide hydrogen.

RATIONALE: The electron capture dissociation (ECD) of proteins/peptides is affected by the nature and sequence of amino acid residues. Electron capture/transfer with no dissociation is an intriguing phenomenon that has occasionally been observed. We have previously identified that diarginated peptides enriched with glutamic acid residues were found to show suppression of backbone fragmentation. In this paper, we report the effect of geometrical parameters of a peptide, including chain length, conformation and amide hydrogen, on the suppression of ECD fragmentation using synthetic model peptides. METHODS: Glycine containing model polypeptides were used to probe the mechanism. Molecular-mechanics was used to obtain the conformation of the precursor ions. The ECD experiments were performed on a Bruker APEX III 4.7 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. RESULTS: Significant decreases in the intensities of the fragment ions were observed for the 23-mer polypeptide with only one E residue. This implied that the E:R ratio was no longer the sole determining factor for the occurrence of suppression effects. Results of conformational searches showed that there was a hydrogen-bonding 'ladder' formed in the 23-mer polypeptide, which was not found in the 15-mer peptide. Substituting the normal amino acid residues by the corresponding N-methylated amino acid residues in the model peptide, the suppression effect disappeared. CONCLUSIONS: Our results indicate that survival of the intact reduced peptide ion after electron capture depends also on the length of the peptide. The amide hydrogen was critical in forming the resonance structure that suppressed the ECD fragmentation.

A role for the sodium pump in H2O2-induced vasorelaxation in porcine isolated coronary arteries.

Hydrogen peroxide (H2O2) has been proposed to act as a factor for endothelium-derived hyperpolarization (EDH) and EDH may act as a 'back up' system to compensate the loss of the NO pathway. Here, the mechanism of action of H2O2 in porcine isolated coronary arteries (PCAs) was investigated. Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (1nM-50µM), a thromboxane A2-mimetic or KCl (60mM). Concentration-response curves to H2O2(1µM-1mM), bradykinin (0.01nM-1µM), sodium nitroprusside (SNP) (10nM-10µM), verapamil (1nM-10µM), KCl (0-20mM) or Ca(2+)-reintroduction (1µM-10mM) were constructed in the presence of various inhibitors. Activity of the Na(+)/K(+)-pump was measured through rubidium-uptake using atomic absorption spectrophotometry. H2O2 caused concentration-dependent vasorelaxations with a maximum relaxation (Rmax) of 100±16% (mean±SEM), pEC50=4.18±0.20 (n=4) which were significantly inhibited by PEG-catalase at 0.1-1.0mM H2O2 (P<0.05). 10mM TEA significantly inhibited the relaxation up to 100µM H2O2 (P<0.05). 60mM K(+) and 500nM ouabain significantly inhibited H2O2-induced vasorelaxation producing a relaxation of 40.8±8.5% (n=5) and 47.5±8.6% (n=6) respectively at 1mM H2O2 (P<0.0001). H2O2-induced vasorelaxation was unaffected by the removal of endothelium, inhibition of NO, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa Kir, KV, KATP or cGMP. 100µM H2O2 had no effects on the KCl-induced vasorelaxation or Ca(2+)-reintroduction contraction. 1mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na(+)/K(+)-pump activity. We have shown that the vascular actions of H2O2 are sensitive to ouabain and high concentrations of H2O2 are able to modulate the Na(+)/K(+)-pump. This may contribute towards its vascular actions.

Clinically important detection of infection as an 'incidental' finding during cancer staging using FDG-PET/CT.

BACKGROUND: FDG-PET/CT is widely used in the management of a variety of malignancies with excellent overall accuracy, despite the potential for false positive results related to infection and inflammation. AIM: As cancer patients can develop clinically inapparent infections, we evaluated the prevalence and nature of incidental findings reported to be suggestive of infections that had been identified during clinical cancer staging with FDG-PET/CT. METHODS: The study involved a retrospective analysis of 60 patients managed primarily at our facility from a total of 121 cases identified as having possible infection on clinical reporting of more than 4500 cancer staging investigations performed during the calendar year of 2008. RESULTS: Occult infections were uncommon overall (≤1%), but most often because of pneumonia (31.6%), upper respiratory tract infections (21.1%) or wound infections (15.8%). Abnormal scans contributed to patients' management in 52.7% of cases. Two out of 13 patients whose scan abnormalities were not investigated further had worsening changes on repeated scan and one of these patients had clinical deterioration. CONCLUSIONS: In patients with FDG-PET/CT scans suggestive of infection and in whom a final diagnosis could be reached, the positive predictive value for FDG-PET/CT scans was 89% suggesting that abnormal scans indicative of infection should be investigated further in this population.

Comparison of clinical with pathological nodal staging from systematic mediastinal lymph node dissection in early resectable non-small cell lung cancer.

INTRODUCTION: We compared the accuracy of clinical nodal (cN) status N0-1 with that of pathological nodal (pN) status obtained from systematic mediastinal lymph node dissection (SMLD) in primary non-small cell lung cancer. METHODS: Data from 22 consecutive patients, who underwent lung cancer resection and SMLD of at least three mediastinal lymph node stations, from November 2001 to May 2003, were ana1ysed retrospectively. Only patients with cN0-1 status on computed tomography (CT) referred for surgery, were included in this study. RESULTS: Mean age of patients was 66.6 +/- 8.1 years with a male to female ratio of 17:5. Mean number of lymph node stations dissected was 5.8 +/- 1.8. 41 percent had squamous cell carcinoma, 45.5 percent had adenocarcinoma, and 4.5 percent each had large cell carcinoma, bronchioalveolar carcinoma or a lymphoepithelial carcinoma. pN2 metastases were found in 27.3 percent of patients. The sensitivity of cN0-1 was only 12.5 percent, with a specificity of 92.9 percent and an area under the receiver operating characteristics curve of 0.53. The positive and negative predictive values of cN0-1 status were 50 percent and 65 percent, respectively, with an accuracy of 59 percent. 41 percent of patients were understaged with 27.3 percent in pathological stage III. Curative resections were achieved in 59 percent of patients. CONCLUSION: The sensitivity of cN0-1 status based on CT alone is extremely poor when compared with pN status from SMLD. Based on cN0-1 status alone without SMLD, 27.3 percent of patients in pN2 would have been understaged. We recommend that all patients with cN0-1 status should undergo SMLD of at least three appropriate mediastinal node stations, for more accurate staging.

Event-related brain correlates of associative learning without awareness.

Controversy exists about whether associative learning occurs without awareness. In an earlier study using subthreshold (subliminal) stimuli, we reported evidence that such learning could occur as measured by event-related brain potentials [ERP; Cons. Cognit. 6 (1997) 519]. In the present study, we extend these findings by changing several aspects of the methodology in order to provide a more stringent test of this effect and to examine its generality. We used two matched words (murder and cancer) as conditional stimuli (CS); a 100 dB white noise blast as unconditional stimulus (US); a CS-US interval of 3 s; and a full-factorial design with CSs counterbalanced. The conditioning-acquisition phase occurred when the CSs were perceptually unconscious, as confirmed by a subsequent behavioral task. The conditioning-acquisition and postconditioning-extinction phases were examined for ERP evidence of associative learning. The clearest and strongest evidence for associative learning without awareness was observed in the ERP component measures (up to 1 s, poststimulus) in the postconditioning-extinction phase. The CS+ was significantly more positive than the CS- in the P3b-LP component region, which is highly consistent with the results of our earlier study. Differences also were observed in the P1-P2 components. In an unexpected finding, we observed a significant positive slow potential shift for the CS+ in the region between 1 and 3 s poststimulus. We discuss these results and their implications for our understanding of associative learning and awareness.

Identification and determination of nucleosides in rat brain microdialysates by liquid chromatography/electrospray tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of brain basal nucleosides (inosine, guanosine and adenosine) in microdialysates from the striatum and cortex of freely moving rats. A microdialysis probe was surgically implanted into the striatum or cortex of individual rats and Ringer's solution was used as the perfusion medium at a flow rate of 0.3 or 0.5 microl/min. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of inosine, guanosine and adenosine was carried out on a cyano column using a mobile phase of 10 mM ammonium acetate, 1% acetic acid and 8% methanol at a flow rate of 0.4 ml/min. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for inosine, guanosine and adenosine was 80, 80 and 40 pg on column, respectively. With this method, the intercellular basal inosine, guanosine and adenosine concentrations in striatum and cortex of rat were determined.

Renal cell carcinoma and malignant phase hypertension.

Many cross-sectional and follow-up studies of large numbers of patients with hypertension have demonstrated an increased prevalence and mortality from renal cancer. We report the details of three patients with renal cell carcinoma from a series of 254 consecutive patients with malignant phase hypertension, an excess over the expected number reported from several large published series with non-malignant hypertension. In view of this excess we investigated the prevalence of hypertension in a series of 192 consecutive patients who presented with a diagnosis of renal cell carcinoma, in comparison with a local unselected population screening survey. Hypertension was found in 43% of the renal carcinoma patients and 20% of the local population, also a clear excess. The mechanism of the association between renal cancer and malignant and non-malignant hypertension is unclear.

Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue.

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.

Resection of large primary chest wall chondrosarcoma with reconstruction: 2 case reports.

We report two cases of large chest wall primary chondrosarcoma, one of the sternum and the other of the lateral chest wall. Both were treated by radical resection and reconstruction using marlex mesh and methyl methacrylate "sandwich" prosthesis and pedicled latissiumus dorsi flap.

Pneumonia presenting as acute abdomen in children: a report of three cases.

From 10th September 1998 till 5th June 1999, the Paediatric and Cardiothoracic Surgery Units of Sultanah Aminah Hospital Johor Bahru managed three children with lung collapse secondary to pneumonia. The dominant initial clinical presentation in all three cases was acute abdominal pain. Basal pneumonia was diagnosed in two cases post-operatively after surgical contributory causes were excluded intra-operatively. Thoracotomy, evacuation of infected debris and decortication of the collapsed lung was done in all three cases. In children presenting with acute abdominal pain, basal pneumonia should be considered as a possible contributory cause.

Reaction of organic nitrate esters and S-nitrosothiols with reduced flavins: a possible mechanism of bioactivation.

Organic nitrate esters, such as glyceryl trinitrate and isosorbide dinitrate, are a class of compounds used to treat a variety of vascular ailments. Their effectiveness relies on their ability to be bioactivated to nitric oxide (NO) which, in turn, relaxes vascular smooth muscle. Although there have been many biological studies that indicate that NO can be formed from organic nitrate esters in a biological environment, the chemical mechanism by which this occurs has yet to be established. Previous studies have implicated both flavins and thiols in organic nitrate ester bioactivation. Thus, we examined the chemical interactions of flavins and thiols with organic nitrate esters as a means of determining the role these species may play in NO production. Based on these studies we concluded that a reasonable chemical mechanism for organic nitrate ester bioactivation involves reduction to the organic nitrite ester followed by conversion to a nitrosothiol. The release of NO from nitrosothiols can occur via a variety of processes including reaction with dihydroflavins and NADH.

Multiple coronary artery bypass grafting in dextrocardia: case report.

This is a case report of an unusual case of a patient with dextrocardia and "situs inversus totalis" who presented with unstable angina. Coronary angiography revealed severe main stem and severe triple vessel coronary artery disease. The patient later underwent successful emergency coronary artery bypass graft surgery. To the authors' knowledge this is the first reported case in Malaysia and also, the first ever report in the literature of multiple vessel coronary artery grafting, including the use of the right internal mammary artery.

Regional tumor oxygen tension: fluorine echo planar imaging of hexafluorobenzene reveals heterogeneity of dynamics.

PURPOSE: Therapeutic success could be enhanced if therapy were tailored to the characteristics of specific tumors. We have been developing novel approaches to measuring tumor oxygen tension in vivo, and recently reported a method based on 19F nuclear magnetic resonance (NMR) spin lattice echo planar imaging (EPI) relaxometry of hexafluorobenzene (HFB). We have now examined the feasibility of monitoring dynamic changes in regional tumor oxygenation in response to respiratory challenge. Preliminary data in one tumor show distinct differences before and subsequent to irradiation. METHODS AND MATERIALS: Dunning prostate adenocarcinoma R3327-AT1 was grown in the form of pedicles on the foreback of male Copenhagen rats. When the tumors reached approximately 1 cm diameter, HFB (40 microl) was administered by direct intratumoral injection deliberately dispersed to interrogate both central and peripheral regions. Local pO2 was determined using pulse burst saturation recovery 19F NMR EPI on the basis of the spin lattice relaxation rate. RESULTS: Interrogation of both central and peripheral regions of tumors showed bimodal distribution for oxygenation, including many voxels with pO2 < 15 torr. Altering the inspired gas to 100% O2 produced significant elevation for regions with initially high pO2 (P < 0.01), but the temporal course of dynamic changes varied for each voxel. Many voxels with low pO2 showed little response. Following irradiation (20 Gy), tumor oxygenation was significantly elevated and remained high for at least 10 h. CONCLUSION: We believe this method provides a valuable new approach to investigate tumor oximetry that may extend our understanding of tumor physiology, and could have prognostic value.

Formation of S-nitrosothiols via direct nucleophilic nitrosation of thiols by peroxynitrite with elimination of hydrogen peroxide.

Peroxynitrite (ONOO-), a potent oxidant formed by reaction of nitric oxide (NO.) with superoxide anion, can activate guanylyl cyclase and is able to induce vasodilation or inhibit platelet aggregation and leukocyte adhesion, via thiol-dependent formation of NO. Reaction of ONOO- with thiols is thought to proceed through formation of a S-nitrothiol (thionitrate; RSNO2) intermediate and yields low levels of S-nitrosothiols (thionitrites; RSNO), both of which are theoretical sources of NO. Kinetic analysis of NO. production after reaction of ONOO- with GSH established that NO. originates exclusively from the thionitrite GSNO. Further mechanistic investigations indicated that GSNO formation by ONOO- does not occur via one-electron oxidation mechanisms. Nitrosation of GSH could theoretically proceed via intermediate formation of the thionitrate GSNO2, which, after rearrangement to the corresponding sulfenyl nitrite (GSONO), can react with GSH to form GSNO and GSOH. However, no evidence for such a mechanism was found in experiments with NO2. or with the stable nitrothiol tert-butylthionitrate. Using high performance liquid chromatography with chemiluminescence detection, formation of H2O2 was observed after reaction of ONOO- with GSH under both aerobic and anaerobic conditions, at levels similar to the yield of GSNO, indicative of a direct nucleophilic nitrosation mechanism with elimination of HOO-. Our results indicate that ONOO- may contribute to S-nitrosation in vivo and that direct nitrosation of thiols or other nucleophilic substrates by ONOO- may represent an important and often overlooked component of NO. biochemistry.

Reaction between S-nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent chemistry.

S-Nitrosothiols have been implicated to play key roles in a variety of physiological processes. The potential physiological importance of S-nitrosothiols prompted us to examine their reaction with thiols. We find that S-nitrosothiols can react with thiols to generate nitroxyl (HNO) and the corresponding disulfide. Further reaction of HNO with the remaining S-nitrosothiol and thiol results in the generation of other species including NO, sulfinamide, and hydroxylamine. Mechanisms are proposed that rationalize the observed products.