AV Actresses | Jav-Porn

48715

Afterwards the relationship between PV-status PVS; a marker how much the actual PV deviated from the ideal PV and outcome was analyzed using cox-proportional modeling in a prospective outcome cohort of CHF-patients Results: Thirty-one CHF-patients were prospectively included in the validation cohort. Diagnosis-to-balloon time DiaTB and door-to-balloon DoTB , defined as time between arrival in the PCI center and first balloon inflation, were correlated to in hospital mortality with correction for differences in baseline risk profile TIMI risk score. Finally, cardiac TNF- a levels were unchanged after 18 weeks Western diet. The infarct white , border zone red and remote area blue were quantified using ImageJ version 1.

vdog | Free download

(84dpvr, DPVR). This movie is about Beautiful Tits, Female Teacher, Variety, Featured Actress, VR Exclusive: VIDEO ON DEMAND: ADULT MOVIES. DPVR 【VR】【60fps版】. 発売日: 収録時間: 86 分. 監督: ドクジー. メーカー: ケイ・エム・プロデュース. レーベル: KMPVR. ジャンル: 単体. Detail information and discussion on "DPVR 【VR】 【60 Fps Version】 Men See And Women See, I See! FREE DOWNLOAD Update Daily "FHD​". 美咲かんな [DPVR] 【VR】 Misaki Kanna - 【60 Fps.. Failure to include FileJoker links for Direct Download posts will result in deletion of. DPVR [VR] [60fps Edition] Whether Viewed By Men, Or Viewed By Women, It's Filled With Plenty Of Download Favorite Correction.

Dpvr-092 ダウンロード. Relation between sst2 reduction during hospitalisation between admission and discharge and mortality left or rehospitalisation right within 1 year.

Download xxx videos bbc anal gape, anal Posted on File name: (​VR) DPVR 男性が見ても女性が見ても、あーなるほど!がいっぱい!イカセ. Watch JAV Dpvr online in the best Japanese porn streaming tube. Best Dpvr Japanese Adult Videos online free HD. DPVR File Download | DPVR Streaming. Photo cover. DPVR Photo Cover. Item ID. DPVR Title. [VR] [60fps Edition] Whether Viewed By Men. DownLoad JAV HDPorn Video, the best uncensored JAPANESE porn videos in DPVR 3D AfestaVR Takumi 匠2+通常3-期間限定発売-もしもスーパー. Japanese Asian 3D VR porn free download, JAV, Japan VR AV, Virtual reality, Japanese adult video, vdog 吹石みゆ, 南瀬奈 若奥様が性の手ほどき!.

Belgian Society of Cardiology 38th BSC Annual Congress - PDF Free Download

Free Download HD FHD DVD WMV MKV MP4 AVI. + Show This Video. Download This Video HERE DPVR Kanna Misaki – VR →. Free Download HD FHD DVD WMV MKV MP4 AVI. + Show Download This Video HERE EXVRA. DPVR Kanna Misaki – VR.Dpvr-092 ダウンロード Free JAV Download-無料JAV動画 FREE DOWNLOAD HERE: [DPVR] 【​VR】 Misaki Kanna – 【60 Fps Version】 Men See And. loading video. Click to Download/Buy DPVR in HD UMSO Jav pop Outdoor Creampie Bath House - I Took My Wife Into The HD Click to Download/Buy DPVR in HD. JAV K M Produce DPVR [VR] Full Length With Bonus Kanna Misaki Turned Into A Love Slut! CLVR, GB (5). CLVR, GB (4) TMAVR, GB (6). TMAVR, GB (3) seoauditing.ru4, GB. seoauditing.ru4, DPVR [VR] An Option To Cum As Many Times As You Like Within The Time Limit Available. Creampie Massage Download Favorite Correction.

Dpvr-092 ダウンロード.

Related videos DPVR · DPVR Mar. 13, 86min. +. [VR] [60fps Edition] Whether Viewed By Men, Or Viewed By Women, It's Filled With Plenty Of Moments That. #=GS D2V5C5_NAEGR/ AC D2V5C #=GS Q4Q1Y1_LEIMA/​ AC Q4Q1Y #=GS Q4E_TRYCC/ AC Q4E #=​GS.

HAY A - Japan VR Porn. Rate: 0; 1; 2; 3; 4; 5. Download video: Download or WATCH ONLINE. seoauditing.ru Download file Free Book PDF hmk latviya buldurskij sovhoz VVIP hmk latviya buldurskij sovhoz tehnikum Pdf Download free Book file PDF Funai Dcvr d Dpvr d Dbvr d Dvd Vcr Service · Chatels Vision A.   Dpvr-092 ダウンロード Exclusive Prestige Actress, Will Lend You download jav vdd Bokep Jav Cl Affair Ogawa Rin xrw /tag/MONE/ /JJBK/ MDVR jav Japanese. Click to Download/Buy DPVR in HD. DPVR JavHD [VR] If An Sasakura Was A Relaxing Men's Esthetician About. Movies Details. Monacoin download + 7,)-$ 3"$,+$)3,$/75/.(!6 $,+$ +!*(,+$ 7+(/.(!*+),+ 2 3"$,+5 * *)30​$35/.6TY 7)=#+2)8.+!)*92,$,+$ / $)/ (-) 3)!)!*3(3,$50 $#@$4(5. Mol Biol Evol ; seoauditing.ru KlQGNAGGAAGAYELDPVRAPMN R I A G T A G G E SN I, D P V R GA WN.

Dpvr-092 ダウンロード

Download seoauditing.ru DPVR Hatano Yui 29yo. GiB x EXVR VR3D 4K Berokiss. GiB. Download Share & Get Free Premium! DPVR B - JAV VR Watch Online. DPVR B - JAV VR CBIKMV B - Watch Online VR · JUVR B​.  Dpvr-092 ダウンロード G8>[*[email protected]^BK_=OK;]'W_05G>P-E^92# @[WQ$ M[?\ [email protected]​FM14D9ITI=F_$:DV%)L;)[4[%K\!N?8[Y?'P;[email protected]<&V M. See note on all banks (–). − − Customers can download cash from their accounts into the card, so Duration = T{1 − [coupon size/(MV × r)]} + [(1 + r)/r][1 − (DPVR/MV)]. ().

[v2] HPPA: i, PS/2 and graphics emulation | Patchew

Download file Free Book PDF garmashev d.l kudryavcev f.a markov a.p Pdf at Fantasies By Cindi Myers · Funai Dpvr H99a1ed Service Manual Symphonic Funai cc Ewc Ssc Color Tv Vcr Combination Repair. Collector`s Selection From The First Half Of ; DPVR | Streaming | Download | #Kanna Misaki DPVR [VR] [60fps Edition] Whether Viewed By Men.  Dpvr-092 ダウンロード Download file Free Book PDF diagram of hydrostatic koxciu goximicroinsurance com Pdf book pdf download diagram of hydrostatic Funai Dpvr Hed Service · Gem E 2plbn Factory Service Work To Creating And Sustaining Authentic Voice And · 92 Toyota Camry Cooling System. Info; Share; Download; Resize; Turn light; 45 Views Similar Videos. [​ChineseSub] UMAD Porn Online [中文字幕]如能隨意控制 HD 49 views. ​ DPVR JavSeen [VR] Long Length/2 Fucks Total Strategies In 5 HD​. YOU SHOULD NOT DOWNLOAD, MAIL, FORWARD, DISTRIBUTE, SEND OR SHARE THE INFORMATION OR DOCUMENTS CONTAINED ON THIS WEBSITE​. nT z??6 g#/: RNL# (pJ0 =l&D 92}n; lb?? (pR?g @} u %N,? >J/B WY-B MaA9 4?a] 3ly"*DpVR H;`?9 z6*rb %gU# 1on) j6=K%?:TQ N|1U `?Kj 9Odq -&+​h3. 

Dpvr-092 ダウンロード. DPVR File Download | DPVR Streaming

DPVR [Vr] [60Fps Version] Whether A Man Or A Woman Sees It! Is Full! I Can Understand The Trick! Onna'S Child Teaches Comfortable Sex! Kanna Misaki. Dako, A) and anti-rabbit-AP Powervision (pure, DPVRAP, Out of 24 patients included in this study, 22 patients (92%) suffered. See note on all banks (–). − 99 Retailers use a terminal to download cash from the card. A number of Duration = T{1 − [coupon size/(MV × r)]} + [(1 + r)/r][1 − (DPVR/MV)]. ().  Dpvr-092 ダウンロード Get Free Download Ebook and Manual Reference. Atlas Terex Excavator Workshop Manual Spanish · Funai Dpvr Dvd Vcr Owners Manual · Haier Abfcbha Repair Service Manual User Guides · Toyota Corolla​. About; Download; Share; Playlist · DPVR Jav Free jav [VR] Full Length &#; 2 Fucks Full Capture With 5 Segments Miyu Saito views 86% [EnglishSub] MIFD Best Jav Porn A 51cm Waist An Asian Slut With. Natakwala lane, Near platform seoauditing.ru No. 8,Borivali West, MUMBAI. - 9. Mumbai. BULLION KA JHARKHAND. GIRIDIH. CANARA BANK Branch in Charge. E Pvr Road Saket Delhi New. Smt. Babita​. Tv series male nude torture New York, New York New York NY seoauditing.ru StartEngine Capital, LLC 6 U&?0*9-$)D9&) 92#M9D​;!&.

Dpvr-092 ダウンロード

The prevalence of this condition amongst hospitalized patients was 0. We excluded patients. Amongst these patients, 84 This prevalence was 3. There was no significant difference between men and women distribution. The prevalence of COPD was higher in the group of pericardial diseases with cancer ratio of 1.

This incidence was 1. Patients with pericardial diseases and cancer were more often readmitted to the hospital ratio of 1. The mortality during hospital stay was significantly higher in the group of patients with pericardial diseases and cancer ratio of 4. It was 7. The mortality during the year following the pericardial event was also greater in the group of patients with pericardial diseases and cancer ratio of 4. Conclusions: The prevalence of cancer amongst patients with pericardial diseases was significantly higher than the prevalence of cancer amongst patients with other cardiovascular diseases as well as amongst the totality of patients hospitalized during the same period.

These patients had also higher prevalence of COPD, which may be linked to the high prevalence of lung cancer. This study shows that the prevalence of cancer amongst patients suffering from pericardial diseases is statistically significant, and that these patients have a very poor prognosis with high mortality and frequent readmissions to the hospital after the pericardial event. However, an increase in stent thrombosis has been observed. A current challenge is thus to develop new bioresorbable stents combining optimised mechanical and biodegradation properties together with limited thrombogenicity.

In this context Fe-based alloys are amongst the good candidates for stent manufacture. In this work blood compatibility of a new Fe-based alloy was studied in vitro via assessment of haemolysis and platelet activation.

Methods: Human whole blood was incubated for 60 minutes with either the Fe-based alloy, pure iron, cobalt-chromium Co-Cr alloy composing the bare metal stent used as a reference or magnesium Mg alloy composing a bioresorbable stent newly on the market.

For platelet activation assays, human washed platelets were incubated for 60 minutes with each alloy before measuring their reactivity to a platelet agonist by flow cytometry, using CD62P and PAC-1 antibodies. In addition, phosphorylation of PKC substrates was evaluated by western blot. Moreover phosphatidylserines were quantified by flow cytometry to evaluate the procoagulant activity of platelets.

Results: None of the alloys induce significant red cells haemolysis. In contrast, Fe-based alloy and pure iron decreased significantly their response to the agonist. Mg based alloy also decreased their response but to a lesser extend Figure 1. A drastic inhibition of the phosphorylation state of PKC substrates was also observed with the Fe-based alloy after activation with thrombin 0. Since similar inhibitory effects were obtained when using a conditioned-reaction medium previously incubated with this Fe-based alloy, we postulate that its biocorrosion might release components counteracting platelet activation.

Finally the Fe-based alloy also decreased significantly the percentage of phosphatidylserine positive platelets after activation with thrombin 0.

Conclusions: The Fe-based resorbable scaffold doesn t induce haemolysis and displays anti-thrombogenic properties. Because stent implantation is currently still associated with thrombosis our stent is a promising platform for next-generation stent technologies. Human platelet activation quantified by PAC-1 following thrombin stimulation. Fe-based alloy inhibits platelet activation. Contrast induced nephropathy is a common clinical problem and it can be prevented by using low contrast volume.

Primary angioplasty was performed in cases. All contrast injections were given by hand. Regular follow-up of the patients was performed at 30 days. The procedures were performed through femoral route only. Pre-dilatation of the lesions with balloons was performed in cases. IVUS was used in only 2 patients. A variety of coronary drug eluting stents from various companies were used in the procedures. Buddy wires were used in 16 cases.

Left main stenting was performed in 17 cases. Diabetes was managed with intermittent doses of insulin at admission and insulin mix regular with NPH insulin at discharge along with oral diabetic medications. Mild reversible contrast induced nephropathy CIN was observed in four patients.

One another patient with creatinine 5. He was put on regular hemodialysis and later he was started on medical management after 1 m. Five mortality was observed in this series, and of these five patients four had cardiogenic shock and one patient expired after discharge due to possible acute stent thrombosis.

Mild cardiac failures were seen in 16 cases, who were treated with frusemide injections and infusions. Two patients required ventilator for congestive heart failure therapy. Coronary perforation and wire breakages were not seen and proximal mild stent edge-dissections was seen in 2 cases, which were treated with additional stents. Dressler s syndrome was seen in two cases and they were managed with pericardiocentesis and low dose steroids.

Groin hematomas requiring one unit of blood transfusion was seen in 3 cases. Conclusions: Angioplasty and stenting could be performed safely in patients with acute coronary syndromes using Cordis diagnostic catheters using low volume of contrast. Low contrast volume usage would result in lower incidence of contrast induced nephropathy and cardiac failures. Echocardiography is the first line imaging technique but there is actually no single RV echocardiographic parameter reliable enough to be universally accepted due to the complex geometrical anatomy of the RV.

Recently, RV 2D speckle tracking has been proposed as a new echocardiographic method for RV evaluation. Competitive risk survival analyses were performed to predict univariate and multivariate parameters for the secondary endpoint. After a median follow-up of 4. In multivariate cox regression, age, ischemic etiology, diabetes, NYHA class III-IV, and beta blocker treatment were independent clinical predictors of overall mortality. Conclusions: RV dysfunction assessed either by cmr or echocardiography predicts CV mortality with significant additional value over baseline clinical and echocardiographic parameters.

Importantly, the predictive value of 2D-RV-GLS was superior to cmr and the other conventional echocardiographic parameters. However, the course of Chagas patients during episodes of decompensated HF has not been sufficiently studied.

Therefore, we sought to investigate the course of Chagas patients during episodes of decompensated HF and during early follow-up after hospital discharge. Methods: We analyzed a prospective cohort of patients admitted with decompensated HF from August through December ; mean age was Main etiologies were dilated cardiomyopathy in During the course of their hospitalization Results: Compared to patients with other etiologies, Chagas patients were more frequently admitted for cardiogenic shock At discharge, Chagas patients had lower sodium level Further, Chagas patients were more likely to receive a transplant during hospitalization After discharge, they were more likely to be readmitted, die or be transplanted at days Figure.

Conclusions: Patients with Chagas cardiomyopathy have a more severe clinical presentation during episodes of decompensated heart failure and greater degree of cardiac remodeling. Prognosis during hospitalization as well as at days after discharge is poorer among Chagas patients. Our findings support specific interventions targeted to this high risk population during episodes of decompensated heart failure and early after hospital discharge.

However, the value of IABC has not been sufficiently explored in patients with chronic heart failure during episodes of acute heart failure and cardiogenic shock HF. We compared the clinical, echocardiographic and hemodynamic data of non-survivors versus transplant patients Table and found that transplanted patients had higher LV ejection fraction Interestingly, variables potentially associated with a poor response to IABC implantation, such as presence of right ventricular dysfunction in echocardiogram or Chagas etiology, did not differ between groups Table.

Conclusions: The use of IABC in patients with cardiogenic shock may be an adequate first step of mechanical support in centers where VADs are not broadly available. We evaluated the influence of increasing age, clinical variables and laboratory tests on the prognosis of patients admitted for heart failure.

Results: The mean age of the patients was Over the period, The rate of events according to the quartiles of age was: Conclusions: Age, ejection fraction, sodium and hemoglobin at hospital admission were the clinical and laboratorial variables associated with higher risk of mortality and readmission in days fallow-up after hospital discharge of patients hospitalized for decompensated heart failure.

However, some recent studies have challenged this association. We sought to explore the influence of renal dysfunction in a comtemporary cohort of patients with decompensated heart failure Methods: Renal dysfunction may occur in up to one-third of patients hospitalized for decompensated heart failure, a finding that has been associated with worse prognosis.

Throughout this period, Mean values of creatinine and urea at admission were respectively 1. Conclusions: In our study renal function was not associated with readmission rates in mortality at days.

This finding further challenge the traditional association between admission renal function and prognosis during episodes of heart failure decompensation and offer alternative information for prognostic interpretation in this scenario. Significant PH leading to right ventricular failure RVF is associated with a worse outcome following surgery. The aims of our study were 1 to characterize the hemodynamic changes following LVAD implantation; 2 to identify predictors of outcome.

Methods: We performed a retrospective, single centre analysis of patients having a LVAD implanted between and who had a baseline and post implant right heart catheterism RHC. The variables of interest were gathered during pre-transplant T 0 and pre T 1 and post T 2 implant examinations. We drew statistical comparisons of biological, ultrasound and hemodynamic variables before and after LVAD implantation.

Furthermore, the incidence and nature of complications in 30 days following LVAD implantation were collected. A Kaplan Meier analysis was used to calculate short and long-term survival post-implantation. Using a linear regression model, the variables of interest were correlated with patients levels of post-implant pulmonary vascular resistance DPVR.

Detailed hemodynamic data are shown in Table 1. Moreover, LVAD implantation improved pulmonary arterial compliance 3. Four patients required temporary right ventricular assist device support.

The linear regression showed that age is inversely correlated to DPVR. A weaker correlation was also found between the latter and the diastolic pulmonary pressure gradient DPG. Conclusions: In our cohort, LVAD implantation resulted in a significant improvement of PH, mainly driven by a normalisation of left-sided filling pressures and improvement in cardiac output. This suggest that PH in end-stage HF with reduced ejection fraction is a purely passive mechanism, associated with a natural aging process, that does not influence outcome.

Therefore, we believe that RVF following LVAD implantation may be explained by another mechanism independent of the pulmonary circulation. However, limited information is available about the mechanism. The mean follow-up of both the incident and antecedent-analysis was days.

There was no impact on atrial-fibrillation burden see Table. This beneficial effect on ventricular arrhythmias might be related to cardiac reverse remodeling. Monitoring PV might offer prognostic information and might be a target for tailored therapy. Methods: The correlation between technetium- 99 Tc -labeled red blood cell measured PV and calculated PV was first determined in a validation cohort. Afterwards the relationship between PV-status PVS; a marker how much the actual PV deviated from the ideal PV and outcome was analyzed using cox-proportional modeling in a prospective outcome cohort of CHF-patients Results: Thirty-one CHF-patients were prospectively included in the validation cohort.

The mean PVS in the outcome cohort was 6. An increase in PV is independently associated with a higher risk for adverse outcome and a slight contraction of the predicted PV seems to be related to less adverse events. Higher dosages of Renin-Angiotensin-Aldosterone blockers are associated with a higher odds for having an optimal PV-status. However, changes in the field of noninvasive diagnostic testing might have an impact on the need for performing an EMB in certain scenarios.

Methods: We performed a retrospective analysis of consecutive EMBs performed in a single, non-academic, center. EMBs were performed between February and March Baseline characteristics including non-invasive imaging and hemodynamic profile were assessed.

Overall one major 1. However, in patients presenting with an unexplained restricted cardiomyopathy, technetium-labeled bone scanning could offer a non-invasive approach to establishing the diagnosis of ATTR, mitigating the need for EMBs in a subset of patients. Circulating soluble ST2 sst2 concentrations increase as a result of cardiomyocyte stress and cardiac fibrosis.

This relatively new and promising biomarker has recently demonstrated to be a strong predictor of mortality in acute heart failure. Purpose: We determined the circulating levels of sst2 in patients with acute heart failure.

We aimed to assess the relation between a single measurement as well as serial measurements with mortality. Levels of sst2 were measured at admission, after 48 hours and at discharge. The primary outcome was all-cause mortality within 1 year after admission. Results: 11 patients died within 1 year after admission. When serial measurements of sst2 were taken into account, patients with greater sst2 reductions between admission and discharge had a better survival.

Conclusions: sst2 is a promising prognostic biomarker for assessment of adverse events in patients with acute heart failure. A single measurement as well as serial measurements could be useful for the prediction of mortality in the patient with acute heart failure. Figure 1 left. Relation between sst2 at discharge and mortality left or rehospitalisation right within 1 year. The division of low sst2 versus high sst2 was based on the median sst2 value. Figure 2 right.

Relation between sst2 reduction during hospitalisation between admission and discharge and mortality left or rehospitalisation right within 1 year. The division of large sst2 reduction versus small sst2 reduction was based on the median sst2 reduction. The aim of this study was to determine circulating levels, clinical and imaging correlates and prognosis value of FGF23 and sst2 in HFpEF. Patients underwent a complete 2D echo and cardiac magnetic resonance cmr to assess ECV, an indirect index of myocardial diffuse fibrosis.

FGF23 and sst2 were also measured at time of enrolment. After adjusting for this preliminary model, the individual inclusion of sst2 c2 to improve: 4. Kaplan Meier curves showed the prognostic according to the different biomarkers Figure. Idarucizumab is a monoclonal antibody fragment that was developed to neutralize the activity of the direct thrombin inhibitor dabigatran.

We describe the experience of dabigatran reversal with idarucizumab in 10 patients undergoing heart transplant surgery at the University Hospitals Leuven. Methods: At the time of listing for heart transplantation, patients requiring anticoagulation because of non-valvular atrial fibrillation, CHA2DS2VASc score 2 and without ventricular assist device or end-stage renal failure, were started on or switched to dabigatran. Upon availability of a donor organ, dabigatran was neutralized with 5 g of intravenous idarucizumab, immediately prior to induction of anaesthesia.

Results: Ten patients have received a heart transplant using this protocol since its implementation at our centre on October 1st, Mean age was Mean time since last intake was 6. Evolution of dabigatran concentration measured by a calibrated diluted thrombin time assay and activated partial thromboplastin time aptt in function of time after idarucizumab administration are represented in Figure 1. All dabigatran concentrations post idarucizumab were unmeasurably low.

Mean aptt reference range s was During surgery, patients received on average 1. Two patients No adverse reactions or unexpected events that could potentially be related to idarucizumab administration were noted. There were no thrombotic complications. Conclusions: This is the largest report describing the use of idarucizumab to normalize coagulation in patients on dabigatran awaiting heart transplantation.

Administration of 5 g of idarucizumab led to a sustained and complete biochemical reversal, without thrombotic complications, and without interfering with heparinization for cardiopulmonary bypass.

However, there still were some bleeding events. The availability of an immediately-acting complete reversal agent makes dabigatran an attractive choice for non-vad patients with non-valvular atrial fibrillation who are listed for heart transplantation.

Ejection fraction was measured during index hospitalization and functional mitral regurgitation fmr was assessed at time of discharge. According to ESC criteria we identified Therefore we speculate that HFrEF pts with persistent severe FMR after recompensation should be followed intensively in a dedicated heart failure clinic.

It is currently speculated that oxidative stress, fibrosis, apoptosis, impaired autophagy and altered Ca handling are important factors in the aetiology of DCM. Recent published data demonstrate that Western diet high sugar combined with high fat diet given to healthy rats mimics the human phenotype of DCM.

Whether inflammation contributes to the development of DCM is unclear. In that context, we performed a longitudinal study, examining progression of DCM phenotype with cardiac inflammation. Cardiac function was evaluated non-invasively via echocardiography and invasively via hemodynamic measurements. An oral glucose tolerance test OGTT was performed to measure glucose and insulin levels.

Cardiac fibrosis and TNF-a levels were investigated in harvest tissue samples. Lipid profile was evaluated from blood samples. Results: Western diet given to healthy rats induces hyperglycaemia, hyperinsulinemia, in association with an altered lipid profile already after 18 weeks diet.

After 18 weeks, left-ventricle LV hypertrophy and increased interstitial fibrosis were observed in diabetic rats and were further worsened after 30 weeks diet. Conventional echocardiographic analysis did not reveal significant changes between groups regarding ejection fraction, fractional shortening, heart rate, cardiac output, end-diastolic and end-systolic volumes after 18 weeks Western diet. However, after 30 weeks, systolic function was observed, characterized by an increased end-systolic volume and a decreased ejection fraction and fractional shortening.

In addition, end-diastolic pressure was significantly increased after already 18 weeks diet. Finally, cardiac TNF- a levels were unchanged after 18 weeks Western diet. Conclusions: After 18 weeks, Western diet induces DCM, characterized by wall hypertrophy and increased cardiac fibrosis. After 30 weeks diet, DCM displays additional systolic dysfunction and is associated with increased cardiac tissue inflammation. Our data suggest that inflammation could be a key factor involved in the switch from diastolic to systolic failure in DCM.

Underlying mechanisms remain to be determined in order to evaluate the importance of reducing cardiac inflammation in diabetic patients. Results: In , e cross-sectionally increased Bonferroni corrected p. The risk of developing LVDD over follow-up 9.

The detection of TAD necessitates the availability of cut-off values for normal aortic diameters. To overcome this problem we previously developed gender and BSA specific formulas for upper limit of normal ULN calculation for both SoV and ascending aorta AA based on established guidelines and applicable in a wide age range. Differences between the various ULN values were obtained by subtracting the ULN calculated with our formulas from those of literature.

The importance of gender becomes apparent above the age of 15y resulting in large scattering in ULN values not accounting for gender above 15 y. For the AA, only one other study was available for comparison, but showed incongruent results with our values due to categorization in small age groups.

Conclusions: ULN values obtained with our previously published formulas correlate well and lie within the range of those calculated with the other available formulas. Gender should be taken into account due to its impact on aortic dimensions from adolescence on. To gain insight into the mechanisms of exercise intolerance, we compared cardiac function during exercise in left ventricular diastolic dysfunction LVDD with precapillary pulmonary hypertension PH patients and healthy controls.

Cardiac output Q and mean pulmonary artery pressure P were estimated by Doppler techniques. Both PH 7. Exercise imaging provides insight into disease mechanisms and uncovers ventricular dysfunction not evident at rest. Actually, the clinical outcome significance of myocardial replacement fibrosis in asymptomatic patients remains controversial.

Other local and systemic factors might contribute to the severity of left ventricular LV remodelling, symptoms and patients outcome. Epicardial Adipose Tissue EAT is a source of several proinflammatory and pro-atherogenic cytokines, which can biologically influence the myocardium and epicardial coronary arteries through paracrine or vasocrine actions. Coronary artery disease and AS share similar disease pathways.

Methods: This study assessed the respective contribution of EAT and myocardial fibrosis to the symptomatic status and outcome of patients with AS. Myocardial fibrosis was not predictive of the outcome in these asymptomatic patients. Conclusions: In patients with AS, symptoms are associated with the degree of obstruction and the severity underlying myocardial adverse remodelling. On the other hand, evaluating EAT volume may allow better identification of patients with asymptomatic AS at higher risk of developing cardiovascular events during follow-up.

Indeed, excess EAT could contribute to the inflammatory burden of AS by producing pro-atherogenic cytokines, which in turn promote the calcification process of aortic valves leaflets, and the progression of valvular obstruction. Global myocardial work during exercise echocardiography and outcome in patients with asymptomatic severe aortic stenosis Raluca Dulgheru CHU Liege Background: The management strategy in asymptomatic patients with severe aortic stenosis AS and preserved left ventricular ejection fraction LVEF is controversial.

Aortic valve replacement AVR is associated with significant morbidity and mortality, while there is a risk for heart failure HF and sudden cardiac death SCD with conservative management. Exercise testing is recommended in asymptomatic AS, but the prognostic value of dynamic changes in mean pressure gradient MPG and LV contractility is still debated.

No differences in baseline demographic and echocardiographic characteristics were found between the 2 groups. Double product DP was also similar. GLS at baseline and during exercise were similar between groups. Conclusions: GMW analysis during exercise could provide additional information for risk stratification in asymptomatic patients with severe AS and preserved LVEF, above and beyond assessment of contractile reserve and GLS analysis at rest and during exercise.

This finding is probably related to the higher sensitivity of the parameter, associated with a relative load independency. We aimed to evaluate the impact on the right ventricle RV of short distances races. Echocardiography was performed at baseline and immediately after the race. The evaluation of RV function was defined by the RV basal diameter and other classical parameters. Myocardial speckle tracking provided RV longitudinal strain.

The RV strain was also significantly alterated No correlation was found between the variation of the strain and the variation of the basal diameter of the RV nor the variation of the classical parameters of the right ventricular function. Conclusions: Alteration of myocardial deformation in right ventricle was observed immediately after an endurance exercise,even in a short distance race.

The long-term effect of these alterations remains unknown. Therefore, we aimed to assess both the stroke volume response during a physiological exercise test and the pulmonary venous atrial function in these patients versus patients with congenitally corrected TGA cctga.

At rest, a stack of retrospective-gated cine images horizontal long-axis plane was acquired for volumetric assessment of the pulmonary venous atrium PVA , and 12 radial long-axis cine images each with a rotation of 15 encompassing the whole PVA for strain analysis. Afterwards, patients performed free breathing bicycle exercise at four exercise stages within the CMR bore using a cycle ergometer with adjustable electronic resistance and a real time CMR acquisition protocol.

Strain analysis was possible in 30 patients. This could be due to rigid baffles and a stiff PVA in the former. Larger series, validation of strain measurements with invasive pressure measurements, and strain measurements during exercise are needed. Patients with kidney failure have more frequent and more severe MAC. Methods: between feb and jan , all the RTR followed in the nephrology department were asked to take part this study and underwent a complete 2D echocardiography and a vascular scanner.

All the echos were reanalysed for MAC. Demographic and clinical characteristics biological data, data related to renal failure and transplant were extracted from the medical file. Follow up data was obtained from nephrology database, contact with referring nephrologist or GP. Mean creat: 1. Figure show the Kaplan meyer survival curve according to time after RT. This emphasizes the importance of the control of cardiovascular risk factor in this population. The echocardiographic grading of MR is challenging and the recommended grading parameters have several important limitations.

We developed and validated a novel echocardiographic parameter to grade MR, the average pixel intensity API method, based on pixel intensity analysis of the Continuous Wave Doppler signal. Purpose: In this study, we assessed the long-term predictive value of the API method on clinical endpoints.

On multivariate Cox regression analysis, the API method was predictive for cardiovascular mortality and this was independent of ejection fraction. Therefore, the API method may be considered for the echocardiographic grading of MR severity in clinical practice. Yet, these factors are strongly associated with functional status and outcome. Multivariate predictors determining improvement in exercise ventricular-arterial coupling following CRT included metrics of residual exercise MR and systolic and diastolic LV-function.

Conclusions: A chronic CRT-effect reverse remodeling , but not an acute CRT-effect resolution of dyssynchrony beneficially influences pulmonary pressures, pulmonary vascular reserve and right ventricular arterial coupling during exercise, with the latter strongly relating to functional status.

These findings are mechanistically linked to left ventricular reverse remodeling with improved interventricular-dependence and a reduction in exercise MR. Methods: Two CRT-cohorts were collected.

First, a prospective imaging cohort of heart failure HF -patients in sinus rhythm and a guideline indication for CRT, to assess the impact of RA-pacing on LA-function, morphology and synchronicity.

Second, a retrospective outcome cohort of all consecutive HF-patients undergoing CRT-implantation between and was collected to assess the relation of RA-pacing with outcome, defined as reverse remodeling 6-months following CRT-implant, occurrence of new onset atrial fibrillation AF and HF-hospitalization and all-cause mortality.

Peak atrial longitudinal strain, reservoir and booster strain-rate but not conduit strain-rate improved after CRT in patients with low pacing burden and worsened in patients with high pacing burden see Figure panel A. A total of patients were included in the outcomecohort.

Conclusions: RA-pacing in CRT patients negatively influences LA-morphology, function and synchronicity, which is associated with worse clinical outcome including diminished LV- reverse remodeling, increased risk for new onset AF, HF-hospitalization and mortality. Strategies reducing RA-pacing burden might be warranted, in order to optimally sustain LA synchronicity and function following CRT-implant.

Its main characteristic is the existence of many prominent ventricular trabeculae and deep intertrabecular recesses, mostly localized in the apex of the left ventricle. Methods: -A retrospective, descriptive and analytical study was conducted on a series of 26 patients diagnosed with ILVNC by echocardiographic and MRI criteria, coming from various areas of Morocco, over a period of nine years -between January 1st, and September 1st, The 26 patients were selected according to the criteria of Oechslin: 1 Absence of coexisting cardiac abnormalities others that below.

Conclusions: The echocardiography remains the tool of reference and the MRI should probably be used more systematically because of its best resolution. This diagnosis implies a family assessment and sometimes genetics which remain non accessible in a Third World country like Morocco, Our series remains conclusive even if the number is restricted. This study investigates the effects of left ventricular diastolic function on outcome in patients with rheumatic mitral stenosis undergoing closed mitral valvotomy.

Methods: This is a single centre two year comparative study consisting of 60 patients. We evaluated preoperative and post operative transthoracic Two D echocardiographic parameters of diastolic function and compared both data to evaluate improvement of diastolic function.

Wilkins score ranged from 6 to Mitral valve area MVA increased from 0. It remains a simple, safe, and effective means of treating mitral stenosis in regions where socioeconomic changes have not yet reduced the incidence of rheumatic heart disease and where resources for its treatment are limited there is still a place for this procedure.

No patient had history of cardiovascular or internal disease. Comprehensive transthoracic echocardiography was performed at baseline and then at 3-month intervals.

Conclusions: Left atrial cardiotoxicity, both isolated and concomitant with LV cardiotoxicity, occurs in a large proportion of breast cancer patients undergoing CHEM. The potential impact of the left atrial cardiotoxicity on clinical outcome needs to be investigated in a long-term prospective study.

The aim of this study was to assess mortality and determine risk factors associated to poor prognosis. Methods: IENAV patients were recruited from 3 databases cardiothoracic surgery, echolab, minimum clinical summary. Patients with IE on other valves than aortic valve, on prosthesis or device were excluded. Demographic and clinical characteristics echocardiographic, biological data, and data related to IE were extracted from the medical file. Follow up data was obtained from surgery database, contact with referring cardiologist or GP.

For each patient, diagnosis of IE was verified according to Duke s modified criteria and the Euroscore and Charslton score were calculated. Mortality at 30 days, 1 year and at the end of follow up was calculated. Emboli, age, diabetes were not related. Kidney failure and staphylococcus remain high risk factors.

Surgery had a favourable impact on survival. All Patients with indication of surgery who were denied for surgery died. We investigated regulation of LAIR-1 leukocyte expression after MI in patients and hypothesized that its absence in a mouse model of MI would increase infarct size and adverse remodelling.

Similarly, no differences were observed in inflammatory cell influx or fibrosis. In conclusion, LAIR-1 expression on monocytes and neutrophils is increased in the acute phase after MI in patients, but the absence of LAIR-1 in mice does not influence infarct size, inflammation, fibrosis or adverse cardiac remodelling.

The most prevalent cause of HF is an acute myocardial infarction MI. Heart failure after MI is caused by adverse left ventricular remodelling 3 , 4. Adverse remodelling is largely depending on infarct size, but also on the quality of cardiac repair, both greatly influenced by the inflammatory response after MI 5 — 8.

Although inflammatory cells are important in the clearance of debris and necrotic tissue after MI, their pro-inflammatory activity is also responsible for a variety of detrimental effects. In the sub acute phase of ischemia-reperfusion injury, neutrophils and monocytes play an important role in the increase in infarct size 5 , 9 , In the chronic phase, activated and pro-inflammatory monocytes and T-lymphocytes increase adverse remodelling, which leads to impaired cardiac function 11 , In both processes, leukocyte activation is key and regulated by integration of signals from activating and inhibitory cell-receptors The majority of immune inhibitory receptors contain intracellular domains that — upon activation — are able to down regulate or inhibit activation signals from stimulating receptors.

Thereby, they increase the threshold for leukocytes to become activated and attenuate pro-inflammatory effects. The transmembrane leukocyte-associated immunoglobulin-like receptor 1 LAIR-1, CD is an inhibitory receptor that is expressed on most cells of the immune system, including natural killer cells, lymphocytes and monocytes Next to this, LAIR-1 is also capable of inhibiting cytokine-mediated signals and it can prevent proliferation and induce apoptosis of human myeloid leukemia cell lines LAIR-1 is activated upon binding of its ligands including collagens or collagen-domain containing proteins such as surfactant protein D 15 , It is counteracted by shedding of its ectodomain sLAIR-1 or by secretion of its antagonist LAIR-2 17 , which is why plasma levels of these molecules were also studied.

Though LAIR-1 is capable of regulating immune cell function by for example inhibition of target cell lysis or attenuation of the cytotoxic activity of effector T cells 18 , 19 , its role in vivo has been scarcely addressed and evidence for its role in inflammation following acute MI is lacking.

Moreover, we studied the effect of LAIR-1 deficiency in experimental MI in mice, measuring inflammation, infarct size, adverse left ventricular remodelling and cardiac function. Exclusion criteria were the presence of a chronic inflammatory disease, autoimmune disorder, pregnancy and trauma or surgery in the last six months. The study conforms to the Declaration of Helsinki. Similarly, blood was drawn from healthy controls and subjected to flow cytometry. Timeline and experimental set-up.

Healthy controls and patients with a first-time MI were included in the current study. Venous blood collection in healthy controls and at 3 days and 6 weeks after MI was used for flow cytometric analyses and to collect plasma a. Mice were either subjected to ischemia-reperfusion injury or to permanent left coronary artery ligation.

In the mice subjected to permanent left coronary artery ligation and sacrificed after 3 days, we performed flow cytometric analyses on various tissues and blood c. In the other mice subjected to left coronary artery ligation, we performed echocardiography at baseline, 7 days and 28 days after MI and performed similar flow cytometric analyses d — lower panel. All animals were genotyped prior to the experimental procedure and animal welfare was monitored daily.

Blinded technicians and observers performed the respective operations, data acquisition and analyses. Sample size calculation for myocardial ischemia reperfusion model was based on infarct size as the primary endpoint.

For the permanent ligation model sample size calculation was based on end systolic volume as the primary endpoint. Cellular influx and collagen content in the ischemic area were defined as secondary outcomes. The experimental set-up and timeline of myocardial ischemia-reperfusion injury is displayed in Fig. All operations were performed in the morning before noon.

In a dedicated mouse operation room, anaesthesia was induced by intraperitoneal i. These anaesthetics were preferred over cardioprotective propofol or volatile anaesthetics e.

The heart was accessed through a left lateral thoracotomy with incision of the pericardium. The left coronary artery was ligated for 30 minutes with an Ethilon suture Ethicon with a section of polyethylene tubing placed over the left coronary artery LCA.

Ischemia was confirmed by bleaching of myocardium and tachycardia. After 30 minutes of ischemia, reperfusion was initiated by releasing the ligation, resulting in tissue colour recurrence. A piece of the suture was left in place to allow for accurate ligature positioning and determination of the ischemic area and the area at risk at termination.

The surgical wounds were closed and subcutaneous atipamezole hydrochloride 3. The evening of the day of operation and every 12 hours thereafter, subcutaneous injection of buprenorphin 0. Twenty-four hours after ischemia-reperfusion injury, mice were euthanized using sodium pentobarbital The LCA was ligated at the same location as it was ligated during index ischemia.

The heart was then explanted and rinsed with 0. The left ventricle LV was dissected and a small piece of gauze was inserted in the left ventricular cavity. After 10 minutes, sections were turned to allow for adequate reagent contact. Then, sections were placed in formalin and photographs of both sides of each tissue section were captured using a SZH10 Olympus Zoom Stereo Microscope and IC Capture software, version 2.

The infarct white , border zone red and remote area blue were quantified using ImageJ version 1. Permanent coronary artery ligation was performed as described above for ischemia-reperfusion injury, but leaving the ligature in place, resulting in a permanent occlusion of the left coronary artery.

The experimental set-up including the timeline of the mice sacrificed after either 3 days or 28 days is shown in Fig. Mice that died after MI were thoroughly inspected for the cause of death.

Deaths within 48 hours after MI were considered due to perioperative complications or direct complication of MI. Of the 6 WT, 5 died due to cardiac rupture and 1 to unkown causes. At baseline, 7 and 28 days after permanent ligation, anaesthesia was induced by inhalation of 2. Echocardiography was used to assess cardiac geometry and function. Heart rate, respiration and rectal temperature were constantly monitored and body temperature was kept between Respiration gating, a 3-dimensional motor and trigger points were used to obtain transversal images of the heart during the expiratory phase, either at the end of systole or the end of diastole.

These images were then used for complete 3D reconstruction of the heart. At the end of the follow-up period, mice were euthanized using sodium pentobarbital Blood was collected through orbital puncture in EDTA tubes. The inferior caval vein was incised and the vascular system was flushed with 5 mL phosphate-buffered saline PBS through right ventricular puncture. The spleen and the mediastinal lymph nodes posterior to the heart were excised and contained in PBS for flow cytometric analyses afterwards.

Then, the heart was explanted and cut in half. One half was dissected further into infarct and remote tissue and used for flow cytometry or snap-frozen in liquid nitrogen. Liquid permanent red was used as an enzyme substrate. The different subtypes of collagen were stained using goat-anti type I Collagen , Southern Biotech, , 0. Neutrophils, T-cells, macrophages and collagens were semi-automatically quantified using digital histology.

Collagen content was quantified in tissue sections stained for picrosirius red and photographed under polarized light, converted to gray scale images and expressed as a percentage of the region of interest i. Technical errors may occur due to poor quality upon sectioning or inferior quality of immunohistochemistry stainings, which makes reliable analysis impossible. Cells were incubated for 30 minutes in the dark at room temperature RT.

Before measurement, cells were washed and erythrocytes were lysed using Optilyse C. Infarct and remote tissue were collected 3 days after MI and cut into small pieces of around 1 mm 2. After washing, residual red blood cells were lysed with erythrocyte-lysis buffer. All samples were measured on a Gallios flow cytometer 10 colour configuration, Beckman Coulter, Marseille, France. Kaluza Analysis Software 1.

The gating strategy is shown in Supplementary Fig. Colorimetric analyses were performed using a spectrophotometer nm. Plasma levels were calculated based on standards. Normally distributed data were compared using a two-tailed paired serial measurements or unpaired t-test separate groups. Non-normally distributed data were compared using a Wilcoxon serial measurements or Mann-Whitney test separate groups. A log-rank Mantel-Cox test was used for survival analysis. For comparison, 20 healthy volunteers were included as controls.

The transmembrane expression of LAIR-1 on monocytes was significantly higher in the acute phase 3 days after MI , compared to the chronic phase Similar to monocytes, also granulocytes showed higher LAIR-1 receptor expression in the acute compared to the chronic phase, but no difference between the chronic phase and healthy controls was observed e.

Reactions commenced by adding 10 units of the enzyme per gram of substrate to a total volume of 1 mL. Samples were taken at 4 and 24 h for analysis. Ltd using a TSKgel Amide column 4. The galactooligosaccharide standard was obtained from Jiangsu Ruiyang Biotech Co. The models of the A.

The amino acid sequence of A. Five significant sequences were revealed, including previously described LacA p-galactosidase A or p-galactosidase E , p-galactosidase B, p-galactosidase B, p-galactosidase B, and p-galactosidase E in the genome Table 2. This agrees with the five sequences originally reported by de Vries and his colleagues [16,17]. The genes encoding five p-galactosidases were cloned and sequenced with the methods described above. The amino acids sequences were deduced based on the nucleotide sequences.

The main results are summarized in Table 2. Of five cloned genes from A. The resulting protein comprised of three additional amino acid residues with six amino acid residues variation in different positions compared to LacB of A. The resulting proteins comprised of six amino acid residues variation. A further attempt to amplify and sequence by redesigning the sense primer and using chromosomal DNA as template also failed data not shown.

Therefore, lacD in A. Database searches with A. The sequences were aligned and subsequently used to generate a phylogenetic tree by program MEGA 4. Interestingly all four LacA, LacB, LacC and LacE proteins identified in this study were significantly different in their amino acid sequences and belonged to different subgroups. Four functional domains were well-conserved in the tertiary structure of fungal p-galactosidases.

They contained many highly-conserved aromatic and hydrophobic residues that locate in members of family GH These highly conserved functional domains, as well as other conserved domains, were also present in most of the fungal p-galactosidases Figure 1. The amino acid sequence known to bind galactose in p-galactosidases was conserved in LacA, LacB, LacC and LacE, implying that these four enzymes contained galactose-binding domains [26].

Moreover, some conservative hydrophobic amino acid residues were contained in p-galactosidases both from A. To improve this protein sequence alignment, 3D protein models were predicted for the catalytic domains of these four p-galactosidases and structurally aligned with crystal structures of AoLacA p-galactosidase from A. Despite the low sequence similarities, the conservative domains of the four 3D putative structures were similar to those of the template Figure 2.

The enzymes were expressed and secreted by P. The hybrid expression plasmids cloned in-frame lacA, lacB, lacC and lacE lacked the region encoding the original signal peptide. The activities with ONPG as substrate in the supernatants were The activities with lactose as substrate in the supernatants were No activity was detectable in the supernatant of GSk. The basic enzyme kinetic parameters of the enzymes were determined using ONPG or lactose as substrate.

The Km value of the A. The temperature profiles of the enzymes differed considerably Table 3. The pH optimum of LacA was between 3. The maximum activity of LacC occurred at pH 3. Due to its lager pH spectrum and higher pH flexibility, LacC will potentially perform better than the other p-galactosidases from A.

The substrate specificity of the four p-galactosidases was examined by in the enzyme assays with lactose, xylan, xyloglucan, pectin, arabinoxylan, maltose and sucrose as natural substrates or with chemically synthesized ONPG. All the p-galactosidases predominantly hydrolyzed ONP-p-D-galactose and lactose.

However, no activity on xylan, xyloglucan, pectin, arabinoxylan, maltose or sucrose was detectable. Galactooligosaccharide formation from lactose by transgalactosylation of the enzymes. All four p-galactosidases could synthesize galactooligosaccharides.

The transgalactosylation was optimal in the acidic conditions pH 3. In this study, we have identified five ORFs from A. The ORFs represented a previously described p-galactosidase LacA [7,13,15] and three other p-galactosidases LacB, LacC and LacE that had been identified [16] but not previously biochemically characterized. We have now cloned and expressed the four genes and characterized the biochemical properties and structures of the gene products. This ORF appears to represent a pseudogene.

These results suggest that the p-galactosidase family of A. Evidence reported by Widmer and Leuba [12] pointed to the possibility of the A. They purified three p-galactosidases and identified them as glycoproteins with molecular weights of , and , isoelectric points of about 4.

These properties are similar to those established in our detailed investigations. However, the variation in the properties of three glycoproteins were proposed to be mainly due to dissimilar carbohydrate contents and related to culture conditions [12]. Subsequently, a p-D-galactosidase of A.

Gel-filtration chromatography and SDS-PAGE analysis showed that its molecular weight was , and ,, respectively, suggesting that it was heavily glycosylated. This p-galactosidase was designated lacA and the gene was cloned and expressed in S.

The transcription of lacA was induced by arabinose, xylose, xylan and pectin [29]. The advent of the genomic era has allowed greater detailed investigation of enzyme properties and has shown that specific enzyme activity is linked to the substrate utilization profiles of the isoenzymes [30,31]. With no surprise, the functional four p-galactosidases of A.

  Relative Posts

One enzyme unit is defined as the quantity of enzyme that catalyzes the liberation of 1 pmol of ortho-nitrophenol from ONPG per min or 1 pmol of glucose from lactose under assay conditions.

Protein concentrations were determined by the method of Bradford [24] with crystalline bovine serum albumin fraction V Serva Feinbiochemica, Germany as reference. For the determination of the effect of cations and EDTA on the enzymatic activity, the enzyme was incubated with 5 mM cation or chemical in pH 5. The p-galactosidase activity was tested with both ONPG and lactose as substrates. The determination of the p-galactosidase activity was carried out at pH5. Michaelis constant Km , and Vmax were calculated with Lineweaver-Burk method using the nonlinear curve fitting function available in the OriginPro 8 software package.

Reactions commenced by adding 10 units of the enzyme per gram of substrate to a total volume of 1 mL. Samples were taken at 4 and 24 h for analysis. Ltd using a TSKgel Amide column 4. The galactooligosaccharide standard was obtained from Jiangsu Ruiyang Biotech Co.

The models of the A. The amino acid sequence of A. Five significant sequences were revealed, including previously described LacA p-galactosidase A or p-galactosidase E , p-galactosidase B, p-galactosidase B, p-galactosidase B, and p-galactosidase E in the genome Table 2. This agrees with the five sequences originally reported by de Vries and his colleagues [16,17]. The genes encoding five p-galactosidases were cloned and sequenced with the methods described above.

The amino acids sequences were deduced based on the nucleotide sequences. The main results are summarized in Table 2. Of five cloned genes from A. The resulting protein comprised of three additional amino acid residues with six amino acid residues variation in different positions compared to LacB of A. The resulting proteins comprised of six amino acid residues variation. A further attempt to amplify and sequence by redesigning the sense primer and using chromosomal DNA as template also failed data not shown.

Therefore, lacD in A. Database searches with A. The sequences were aligned and subsequently used to generate a phylogenetic tree by program MEGA 4. Interestingly all four LacA, LacB, LacC and LacE proteins identified in this study were significantly different in their amino acid sequences and belonged to different subgroups.

Four functional domains were well-conserved in the tertiary structure of fungal p-galactosidases. They contained many highly-conserved aromatic and hydrophobic residues that locate in members of family GH These highly conserved functional domains, as well as other conserved domains, were also present in most of the fungal p-galactosidases Figure 1.

The amino acid sequence known to bind galactose in p-galactosidases was conserved in LacA, LacB, LacC and LacE, implying that these four enzymes contained galactose-binding domains [26]. Moreover, some conservative hydrophobic amino acid residues were contained in p-galactosidases both from A.

To improve this protein sequence alignment, 3D protein models were predicted for the catalytic domains of these four p-galactosidases and structurally aligned with crystal structures of AoLacA p-galactosidase from A. Despite the low sequence similarities, the conservative domains of the four 3D putative structures were similar to those of the template Figure 2.

The enzymes were expressed and secreted by P. The hybrid expression plasmids cloned in-frame lacA, lacB, lacC and lacE lacked the region encoding the original signal peptide. The activities with ONPG as substrate in the supernatants were The activities with lactose as substrate in the supernatants were No activity was detectable in the supernatant of GSk.

The basic enzyme kinetic parameters of the enzymes were determined using ONPG or lactose as substrate. The Km value of the A. The temperature profiles of the enzymes differed considerably Table 3.

The pH optimum of LacA was between 3. The maximum activity of LacC occurred at pH 3. Due to its lager pH spectrum and higher pH flexibility, LacC will potentially perform better than the other p-galactosidases from A. The substrate specificity of the four p-galactosidases was examined by in the enzyme assays with lactose, xylan, xyloglucan, pectin, arabinoxylan, maltose and sucrose as natural substrates or with chemically synthesized ONPG. All the p-galactosidases predominantly hydrolyzed ONP-p-D-galactose and lactose.

However, no activity on xylan, xyloglucan, pectin, arabinoxylan, maltose or sucrose was detectable. Galactooligosaccharide formation from lactose by transgalactosylation of the enzymes. All four p-galactosidases could synthesize galactooligosaccharides. The transgalactosylation was optimal in the acidic conditions pH 3. In this study, we have identified five ORFs from A.

The ORFs represented a previously described p-galactosidase LacA [7,13,15] and three other p-galactosidases LacB, LacC and LacE that had been identified [16] but not previously biochemically characterized.

We have now cloned and expressed the four genes and characterized the biochemical properties and structures of the gene products. This ORF appears to represent a pseudogene. These results suggest that the p-galactosidase family of A. Evidence reported by Widmer and Leuba [12] pointed to the possibility of the A. They purified three p-galactosidases and identified them as glycoproteins with molecular weights of , and , isoelectric points of about 4.

These properties are similar to those established in our detailed investigations. However, the variation in the properties of three glycoproteins were proposed to be mainly due to dissimilar carbohydrate contents and related to culture conditions [12]. Subsequently, a p-D-galactosidase of A. Gel-filtration chromatography and SDS-PAGE analysis showed that its molecular weight was , and ,, respectively, suggesting that it was heavily glycosylated.

This p-galactosidase was designated lacA and the gene was cloned and expressed in S. The transcription of lacA was induced by arabinose, xylose, xylan and pectin [29]. The advent of the genomic era has allowed greater detailed investigation of enzyme properties and has shown that specific enzyme activity is linked to the substrate utilization profiles of the isoenzymes [30,31].

With no surprise, the functional four p-galactosidases of A. This might explain the multiple form pattern of the A. Many potential applications of these enzymes have been identified.

Two are particularly attractive: 1 hydrolysis of lactose in dairy products or administration as a digestive supplement to prevent or treat lactose intolerance [32] and 2 transglycosylation of lactose to produce the valuable prebiotic galactooligosaccharide.

All four P-galactosidases hydrolyzed and transgalactosylated lactose but with unique kinetic properties. In many cases, similar pH optima are reported for galactooligosaccharide synthesis and galactoside hydrolysis [18,33].

In contrast, the A. These enzymes have been reported to be useful in the modification of the glycosyl side chains on xylan, pectin, xyloglucan and arabinoxylan amonst other polymers [29]. The potential of A. In addition to the known LacA in A. Anal Biochem ; P-galactosidase from Aspergillus oryzae.

Enzyme Microb Technol ; Current state and perspectives. Appl Microbiol Biotechnol ; Cell ; Biotechnol Adv ; Appl Biochem Biotechnol ; Biotechnol Prog ; Carbohydr Res ; Glycoprotein enzymes secreted by Aspergillus fumigatus: Purification and properties of beta-glucosidase.

J Biol Chem ; Separation and characterization of three multiple forms. Eur J Biochem ; Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate. Michaelis constant Km , and Vmax were calculated with Lineweaver-Burk method using the nonlinear curve fitting function available in the OriginPro 8 software package. Reactions commenced by adding 10 units of the enzyme per gram of substrate to a total volume of 1 mL.

Samples were taken at 4 and 24 h for analysis. Ltd using a TSKgel Amide column 4. The galactooligosaccharide standard was obtained from Jiangsu Ruiyang Biotech Co. The models of the A. The amino acid sequence of A. Five significant sequences were revealed, including previously described LacA p-galactosidase A or p-galactosidase E , p-galactosidase B, p-galactosidase B, p-galactosidase B, and p-galactosidase E in the genome Table 2.

This agrees with the five sequences originally reported by de Vries and his colleagues [16,17]. The genes encoding five p-galactosidases were cloned and sequenced with the methods described above. The amino acids sequences were deduced based on the nucleotide sequences. The main results are summarized in Table 2. Of five cloned genes from A.

The resulting protein comprised of three additional amino acid residues with six amino acid residues variation in different positions compared to LacB of A. The resulting proteins comprised of six amino acid residues variation. A further attempt to amplify and sequence by redesigning the sense primer and using chromosomal DNA as template also failed data not shown. Therefore, lacD in A. Database searches with A. The sequences were aligned and subsequently used to generate a phylogenetic tree by program MEGA 4.

Interestingly all four LacA, LacB, LacC and LacE proteins identified in this study were significantly different in their amino acid sequences and belonged to different subgroups. Four functional domains were well-conserved in the tertiary structure of fungal p-galactosidases. They contained many highly-conserved aromatic and hydrophobic residues that locate in members of family GH These highly conserved functional domains, as well as other conserved domains, were also present in most of the fungal p-galactosidases Figure 1.

The amino acid sequence known to bind galactose in p-galactosidases was conserved in LacA, LacB, LacC and LacE, implying that these four enzymes contained galactose-binding domains [26]. Moreover, some conservative hydrophobic amino acid residues were contained in p-galactosidases both from A. To improve this protein sequence alignment, 3D protein models were predicted for the catalytic domains of these four p-galactosidases and structurally aligned with crystal structures of AoLacA p-galactosidase from A.

Despite the low sequence similarities, the conservative domains of the four 3D putative structures were similar to those of the template Figure 2.

The enzymes were expressed and secreted by P. The hybrid expression plasmids cloned in-frame lacA, lacB, lacC and lacE lacked the region encoding the original signal peptide. The activities with ONPG as substrate in the supernatants were The activities with lactose as substrate in the supernatants were No activity was detectable in the supernatant of GSk.

The basic enzyme kinetic parameters of the enzymes were determined using ONPG or lactose as substrate. The Km value of the A. The temperature profiles of the enzymes differed considerably Table 3.

The pH optimum of LacA was between 3. The maximum activity of LacC occurred at pH 3. Due to its lager pH spectrum and higher pH flexibility, LacC will potentially perform better than the other p-galactosidases from A. The substrate specificity of the four p-galactosidases was examined by in the enzyme assays with lactose, xylan, xyloglucan, pectin, arabinoxylan, maltose and sucrose as natural substrates or with chemically synthesized ONPG.

All the p-galactosidases predominantly hydrolyzed ONP-p-D-galactose and lactose. However, no activity on xylan, xyloglucan, pectin, arabinoxylan, maltose or sucrose was detectable. Galactooligosaccharide formation from lactose by transgalactosylation of the enzymes. All four p-galactosidases could synthesize galactooligosaccharides. The transgalactosylation was optimal in the acidic conditions pH 3. In this study, we have identified five ORFs from A. The ORFs represented a previously described p-galactosidase LacA [7,13,15] and three other p-galactosidases LacB, LacC and LacE that had been identified [16] but not previously biochemically characterized.

We have now cloned and expressed the four genes and characterized the biochemical properties and structures of the gene products. This ORF appears to represent a pseudogene. These results suggest that the p-galactosidase family of A. Evidence reported by Widmer and Leuba [12] pointed to the possibility of the A. They purified three p-galactosidases and identified them as glycoproteins with molecular weights of , and , isoelectric points of about 4. These properties are similar to those established in our detailed investigations.

However, the variation in the properties of three glycoproteins were proposed to be mainly due to dissimilar carbohydrate contents and related to culture conditions [12]. Subsequently, a p-D-galactosidase of A. Gel-filtration chromatography and SDS-PAGE analysis showed that its molecular weight was , and ,, respectively, suggesting that it was heavily glycosylated. This p-galactosidase was designated lacA and the gene was cloned and expressed in S.

The transcription of lacA was induced by arabinose, xylose, xylan and pectin [29]. The advent of the genomic era has allowed greater detailed investigation of enzyme properties and has shown that specific enzyme activity is linked to the substrate utilization profiles of the isoenzymes [30,31]. With no surprise, the functional four p-galactosidases of A. This might explain the multiple form pattern of the A.

Many potential applications of these enzymes have been identified. Two are particularly attractive: 1 hydrolysis of lactose in dairy products or administration as a digestive supplement to prevent or treat lactose intolerance [32] and 2 transglycosylation of lactose to produce the valuable prebiotic galactooligosaccharide.

All four P-galactosidases hydrolyzed and transgalactosylated lactose but with unique kinetic properties. In many cases, similar pH optima are reported for galactooligosaccharide synthesis and galactoside hydrolysis [18,33]. In contrast, the A. These enzymes have been reported to be useful in the modification of the glycosyl side chains on xylan, pectin, xyloglucan and arabinoxylan amonst other polymers [29]. The potential of A. In addition to the known LacA in A.

Anal Biochem ; P-galactosidase from Aspergillus oryzae. Enzyme Microb Technol ; Current state and perspectives. Appl Microbiol Biotechnol ; Cell ; Biotechnol Adv ; Appl Biochem Biotechnol ; Biotechnol Prog ; Carbohydr Res ; Glycoprotein enzymes secreted by Aspergillus fumigatus: Purification and properties of beta-glucosidase.

J Biol Chem ; Separation and characterization of three multiple forms. Eur J Biochem ; Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate. Biotechnology NY ; J Biosci Bioeng ; Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae. Fungal Genet Biol ; The value of genome sequences in the rapid identification of novel genes encoding specific plant cell wall degrading enzymes.

6v8Apd39Vn/fTXQ6bSGVhjrH2OMBoYWc/56av+lXf1Wf9+ DPVr/f8Ab6lX​+Fr/AJtJTbSVTqfUK+m4NmZa1zRtbyS5wrb/1Xv8A5Cr3dexaWS9lj3tsNFld. Get Free Download Ebook and Manual Reference. Frigidaire 36 Repair Service Manual User Guides · Funai Dpvr j Dvd Vcr Service Manual Honda Motorcycle Chf50p Metropolitan Service Shop Manual · Panasonic Pt​.