Cell-specific Dynamic Clamp analysis of the role of funny If current in cardiac pacemaking.

We used the Dynamic Clamp technique for i) comparative validation of conflicting computational models of the hyperpolarization-activated funny current, If, and ii) quantification of the role of If in mediating autonomic modulation of heart rate. Experimental protocols based on the injection of a real-time recalculated synthetic If current in sinoatrial rabbit cells were developed. Preliminary results of experiments mimicking the autonomic modulation of If demonstrated the need for a customization procedure to compensate for cellular heterogeneity. For this reason, we used a cell-specific approach, scaling the maximal conductance of the injected current based on the cell's spontaneous firing rate. The pacemaking rate, which was significantly reduced after application of Ivabradine, was restored by the injection of synthetic current based on the Severi-DiFrancesco formulation, while the injection of synthetic current based on the Maltsev-Lakatta formulation did not produce any significant variation. A positive virtual shift of the If activation curve, mimicking the Isoprenaline effects, led to a significant increase in pacemaking rate (+17.3 ± 6.7%, p < 0.01), although of lower magnitude than that induced by real Isoprenaline (+45.0 ± 26.1%). Similarly, a negative virtual shift of the activation curve significantly lowered the pacemaking rate (-11.8 ± 1.9%, p < 0.001), as did the application of real Acetylcholine (-20.5 ± 5.1%). The Dynamic Clamp approach, applied to the If study in cardiomyocytes for the first time and rate-adapted to manage intercellular variability, indicated that: i) the quantitative description of the If current in the Severi-DiFrancesco model accurately reproduces the effects of the real current on rabbit sinoatrial cell pacemaking rate and ii) a significant portion (50-60%) of the physiological autonomic rate modulation is due to the shift of the If activation curve.

Laparoscopic surgical staging of endometrial cancer: does obesity influence feasibility and perioperative outcome?

AIM: Laparoscopic treatment of early-stage endometrial cancer is the gold standard to reduce perioperative morbidity. Obesity is a well-known risk factor for endometrial cancer and anesthesiological and surgical complications. The authors' aim was to examine the effect of body mass index (BMI) on perioperative parameters and complications in laparoscopically-treated patients with endometrial cancer. MATERIALS AND METHODS: A consecutive series of patients affected by endometrial cancer and their demographic and clinicopathological data were collected. Patients were divided in 41 non-obese (BMI or= 30) groups. All patients had been preoperatively evaluated with hysteroscopic procedures and toraco-abdominal computed tomography (CT) and had been submitted to laparoscopic radical hysterectomy according to Querleu-Morrow, pelvic lymphadenectomy, peritoneal washing, and bilateral adnexectomy. RESULTS: There was no statistically significant difference in blood loss, number of lymph nodes removed, and hospital stay between the groups, but there was a trend towards a lengthening of surgical time in the obese women. There were no major intraoperative and postoperative complications. DISCUSSION: This study demonstrates that laparoscopic approach is feasible and safe in obese women evaluating the anesthesiological risk.

Atypical endometrial lesions: hysteroscopic resection as an alternative to hysterectomy.

BACKGROUND: Endometrial hyperplasia is a precursor to endometrial carcinoma: the risk of progression to invasive endometrial cancer is increased in postmenopausal women and much more in cases of atypical endometrial hyperplasia (25%-30%). In addition, in 12.7% to 42.6% of cases according to various studies, endometrial cancer coexists in patients with diagnosis of atypical endometrial hyperplasia. The aim of this study was to evaluate the correlation between radical hysteroscopic resection of atypical endometrial lesions and the histopathological examination of the uterus. MATERIALS AND METHODS: The authors collected 25 patients referring to the Department of Woman and Child Health, in the University of Padua (Italy) from January 2008 to June 2012, undergoing hysteroscopic resection for atypical polyps and focal atypical endometrial hyperplasia, and following hysterectomy within 30 days. Average age, menopausal status, hormone replacement therapy, body mass index (BMI), presence of hypertension and diabetes, and taking tamoxifen were reported. RESULTS: After hysteroscopic resection in all patients atypical polyps and focal endometrial hyperplasia were confirmed. The hystopathologic evaluation of the uterus reported: in only two (8%) cases, the persistence of atypical endometrial lesion, whereas in 23 (92%) cases the endometrial tissue was negative for atypia or malignancy. CONCLUSIONS: Radical endometrial resection by hysteroscopy may serve as an alternative to hysterectomy in selected patients with atypical focal endometrial lesions, not only in fertile women, but also in patients who refuse hysterectomy or present high anesthesiologic and surgical risks, regardless of the risk of recurrence, and with the necessity of undergoing hysteroscopic close follow-up.

The trend of VEGF-A and PlGF in pregnant patients: a perspective case-control study on 214 women.

OBJECTIVE: The aim of this study was to measure plasmatic concentrations of vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PIGF) in pregnant women, and to evaluate their relationship with age, hormonal status, gestational age, and different diseases of pregnancy. METHODS: We selected a control group of 163 patients (96 fertile and 67 in menopause) and a group of 214 pregnant patients during the whole gestational period. VEGF-A and PlGF were assayed by ELISA and EIA methods, respectively. Statistical analysis was performed using the Mann-Whitney test. RESULTS: The control group showed mean VEGF-A and PlGF values of 89.87 pg/ml and 10.22 pg/ml, respectively; PlGF showed the highest values in menopausal patients. The group of pregnant patients showed VEGF-A values of 27.05 pg/ml and PlGF values of 231.36 pg/ml respectively, with lower (for the VEGF-A) and higher (for the PlGF) statistical significance. These values were not influenced by biological age, but were related to gestational age: VEGF-A showed a decrease and PlGF an increase particularly after the 20th gestational week. PlGF showed a statistically significant decrease compared to physiological gestation in spontaneous and threatened abortions (p < 0.0001) and in ectopic pregnancies (p < 0.0001), an increase in ultrasound and CTG alterations (p < 0.05), and threatened premature delivery and uterine hypercontractility (p < 0.01); on the other hand VEGF-A showed a statistically significant increase in ectopic pregnancies (p < 0.05). CONCLUSIONS: VEGF-A and PlGF may play a diagnostic and prognostic role in pregnancy. Further studies are required to better understand the meaning of variability of their values.

[Surgical options in the treatment of perianal Crohn's disease]. / Opzioni chirurgiche nel trattamento della malattia di Crohn a localizzazione perianale.

INTRODUCTION: About 40% of patients with Crohns disease (CD) have a perianal involvement. Despite the recent introduction of anti-TNF antibody, this therapy has uncertain long-term results and surgery still remains a major treatment option. AIMS & METHODS: This study relates our experience in surgical management of perianal CD without anti-TNF treatment. From July 92 to February 02, 37 patients with perianal Crohns disease were treated, 43 underwent local operations or faecal diversion for fistulas and/or abscesses. Patients not requiring surgery or in therapy with anti-TNF. were excluded from the study. We analysed the outcome of surgical treatment for perianal CD. RESULTS: Male to female ratio was 1:0.6, median age was 36,9 years (range 17-62). Perianal disease included 32 fistulas (16 trans-sphincteric, 2 superficial, 2 ano-vaginal, 10 multiple and complex, 2 horseshoe) and 7 abscesses (5 perianal, 2 ischio-rectal). Local surgery included 1 abscess drainage, 5 abscess drainage and fistula incision with seton insertion, 2 fistulotomy, 9 partial fistulectomy and seton insertion. At surgery, 40% of patients were ongoing a medical treatment with 5-ASA and/or antibiotics, 40% with steroids and/or immunosoppressors, 15% only with 5-ASA and 5% no ongoing treatment. The horseshoe fistulas were managed with a fistulotomy and seton insertion. One patient with ano-vaginal fistula required proctectomy and the other one total proctocolectomy. Patients treated by diverting colonostomy (3) had fistula recurrence after its closure in 100%. 20% of patients required total proctocolectomy and ileostomy for extensive intestinal disease. Of the 27 patients undergoing seton insertion or fistulotomy none had faecal incontinence due to the operation and 38% had a 1 year recurrence. CONCLUSIONS: Perianal CD is a heterogeneous entity, therefore its management is still controversial. Moreover, a high percentage of patients (18% in our series) requires a major surgery due to the extension and seriousness of rectal involvement. In our survey only 12 patients (39%), with trans-sphincteric fistula, could have been theoretically treated with anti-TNF. We wonder if the cost-and-benefit of this medical treatment justifies its application on patients that could undergo a surgical treatment with good long-term results.

Accurate prediction of the bound conformation of galanthamine in the active site of Torpedo californica acetylcholinesterase using molecular docking.

The alkaloid (-)-galanthamine is known to produce significant improvement of cognitive performances in patients with the Alzheimer's disease. Its mechanism of action involves competitive and reversible inhibition of acetylcholinesterase (AChE). Herein, we correctly predict the orientation and conformation of the galanthamine molecule in the active site of AChE from Torpedo californica (TcAChE) using a combination of rigid docking and flexible geometry optimization with a molecular mechanics force field. The quality of the predicted model is remarkable, as indicated by the value of the RMS deviation of approximately 0.5A when compared with the crystal structure of the TcAChE-galanthamine complex. A molecular model of the complex between TcAChE and a galanthamine derivative, SPH1107, with a long chain substituent on the nitrogen has been generated as well. The side chain of this ligand is predicted to extend along the enzyme active site gorge from the anionic subsite, at the bottom, to the peripheral anionic site, at the top. The docking procedure described in this paper can be applied to produce models of ligand-receptor complexes for AChE and other macromolecular targets of drug design.

Three-dimensional structure of a complex of galanthamine (Nivalin) with acetylcholinesterase from Torpedo californica: implications for the design of new anti-Alzheimer drugs.

The 3D structure of a complex of the anti-Alzheimer drug galanthamine with Torpedo californica acetylcholinesterase is reported. Galanthamine, a tertiary alkaloid extracted from several species of Amarylidacae, is so far the only drug that shows a dual activity, being both an acetylcholinesterase inhibitor and an allosteric potentiator of the nicotinic response induced by acetylcholine and competitive agonists. The X-ray structure, at 2.5A resolution, shows an unexpected orientation of the ligand within the active site, as well as unusual protein-ligand interactions. The inhibitor binds at the base of the active site gorge, interacting with both the acyl-binding pocket and the principal quaternary ammonium-binding site. However, the tertiary amine group of galanthamine does not directly interact with Trp84. A docking study using the program AUTODOCK correctly predicts the orientation of galanthamine in the active site. The docked lowest-energy structure has a root mean square deviation of 0.5A with respect to the corresponding crystal structure of the complex. The observed binding mode explains the affinities of a series of structural analogs of galanthamine and provides a rational basis for structure-based drug design of synthetic derivatives with improved pharmacological properties. Proteins 2001;42:182-191.

"Back door" opening implied by the crystal structure of a carbamoylated acetylcholinesterase.

The crystal structure of Torpedo californica (Tc) acetylcholinesterase (AChE) carbamoylated by the physostigmine analogue 8-(cis-2,6-dimethylmorpholino)octylcarbamoyleseroline (MF268) is reported at 2.7 A resolution. In the X-ray structure, the dimethylmorpholinooctylcarbamic moiety of MF268 is covalently bound to the catalytic serine, which is located at the bottom of a long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. Surprisingly, the relatively bulky eseroline is not found in the crystal structure, thus implying the existence of an alternative route for its clearance. This represents indirect evidence that a "back door" opening may occur and shows that the release of products via a "back door" is a likely alternative for this enzyme. However, its relevance as far as the mechanism of substrate hydrolysis is concerned needs to be established. This study suggests that the use of properly designed acylating inhibitors, which can block the entrance of catalytic sites, may be exploited as a general approach for investigating the existence of "back doors" for the clearance of products.

Inhibition of human leukocyte elastase by chemically and naturally oversulfated galactosaminoglycans.

Several samples of oversulfated chondroitin and dermatan were obtained by chemical sulfation and by SAX-HPLC enrichment. The starting products and oversulfated products were tested as potential inhibitors of human leukocyte elastase, an enzyme hypothesized to be involved in the etiology of diseases such as emphysema, atherosclerosis, and rheumatoid arthritis. Chemical oversulfation (SO3H/COOH 1.6-3.2), preferentially occurring at C-6 of galactosamine residues, was found generally to increase the inhibitory power on elastase. Chemically oversulfated galactosaminoglycans thus have potential as therapeutic agents, considering that they produce non-significant effects on the hemocoagulative system. Two naturally oversulfated dermatans sulfate (SO3H/COOH ca. 1.2), mainly oversulfated at C-2 of iduronic acid residues, showed comparatively higher anticoagulant activity (in the HC-II mediated thrombin inhibition test).

Structure-activity relationships in open ansa-chain rifamycin S derivatives as inhibitors of HIV-1 reverse transcriptase.

Three types of open ansa-chain rifamycin S derivatives have been prepared: derivatives with the ansa-chain open at C(29) and the original dihydrofuranone ring; derivatives with the ansa-chain open at C(29) and a furane ring; derivatives with the ansa-chain at open NH-C(15). Only derivatives of the first type are weak inhibitors of HIV-1 reverse transcriptase (IC50 ca.300 microM) while derivatives of the two other types are inactive. It has been hypothesized that the active derivatives inhibit the viral enzyme interacting through the groups C(14)H3, C(13)H3, and C(1)O at the same site as the well-known inhibitors TIBO and Nevirapine. In particular C(13)H3 must be unhindered and in an appropriate position out of the plane containing the chromophore-rings. The open ansa-chain seems to play the role of a lipophylic substituent.

Rifamycins as inhibitors of retroviral reverse transcriptase from M-MuLV, RAV-2, and HIV-1.

29 Rifamycins were tested for inhibition of Reverse Transcriptase (RT) as potential anti HIV drugs. Two purified commercial enzymes from M-MuLV and RAV-2 were used. Anti-RT activity was also measured on a crude lysate of HIV-1. The results show that some derivatives have interesting levels of activity on isolated M-MuLV and RAV-2 RTs, while they are less active on the RT in the crude HIV-1 lysate. The active derivatives include oximes and hydrazones, alkylaminoderivatives, open ansa-chain derivatives and derivatives carrying a modified nucleoside.

Quinolinehydrazones as inhibitors of retroviral reverse transcriptase.

The inhibitory activity of a series of 2- and 4-quinolinehydrazones on retroviral reverse transcriptase has been studied on enzymes from M-MuLV, RAV-2, and on a crude lysate of HIV-1, assuming the first two enzymes as potential models of the third. The highest activity is mainly found in lipophilic, water soluble 4-quinolinehydrazones. The inhibitory activity of these compounds decreases in changing from the M-MuLV to the RAV-2, and HIV-1 enzymes, in this order.

Chondroprotective action of chondroitin sulfate. Competitive action of chondroitin sulfate on the digestion of hyaluronan by bovine testicular hyaluronidase.

Lysosomal hyaluronidase is responsible for the degradation of hyaluronan, a component of the extracellular matrix, in degenerative disorders of the joints. It has been hypothesized that the administration of chondroitin sulfate (both a component of the extracellular matrix and a substrate for hyaluronidase) could compete for this enzyme and reduce the degradation process. The present study shows that a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate is a good competitor of hyaluronan for hyaluronidase. The digestion of hyaluronan is reduced in proportion to the amount of competing chondroitin. The competitive ability is dependent on the 4-sulfate, 6-sulfate composition of the chondroitin mixture. Mixtures richer in the 4-sulfate isomer are more effective. The enzymatic reactions have been monitored by HPLC and PAGE.

Metabolic chiral inversion of anti-inflammatory 2-arylpropionates: lack of reaction in liver homogenates, and study of methine proton acidity.

1. Enrichment in the (S)-enantiomers for (R)-flurbiprofen, (R)-naproxen, (R)-suprofen and (R;S)-ibuprofen was investigated in various subcellular hepatic preparations containing coenzyme A. While such preparations were able to form hippuric acid from benzoic acid, the chiral inversion was never seen. 2. Using 2-dimethylaminoethanethiol 2-phenylpropionate (DEPP) as a model acyl thioester, the acidity of the methine proton was investigated by monitoring the proton/deuterium exchange occurring in deuterated solvents using high-resolution n.m.r. The compound was inert up to 22 h in D2O at 37 degrees C and pD 7.4. In pure methanol or a methanol-water mixture, only solvolysis was seen. In contrast, competitive hydrolysis (k = 0.005 h-1) and proton/deuterium exchange (k = 0.09 h-1) were seen in a CD3CN/D2O (50:50) mixture at 37 degrees C. 3. It is speculated that the failure to characterize chiral inversion of 2-arylpropionates in subcellular preparations may be due to the absence of a microenvironment of adequately moderate polarity.

Comparative study of serum pancreatic isoamylase, lipase, and trypsin-like immunoreactivity in pancreatic disease.

Serum total amylase, pancreatic and salivary isoamylase, lipase and trypsin-like immunoreactivity (TLI) were measured in 16 patients with acute pancreatitis, 37 patients with chronic pancreatitis, 11 patients with pancreatic cancer, and 53 control subjects in order to evaluate the relative value of these tests in the diagnosis of pancreatic disease. In acute pancreatitis patients studied within 2 days from the onset of pain all pancreatic enzymes were abnormally high. In chronic pancreatitis patients serum pancreatic isoamylase and TLI were abnormally low in 8 out of 10 patients with severely impaired pancreatic exocrine function, while lipase was abnormally low in 6 patients. During acute exacerbations of the disease elevated levels of pancreatic isoamylase and lipase, but not of TLI, were found in about one third of cases. In patients with pancreatic cancer the pattern of changes in serum pancreatic enzymes was variable since levels within, below and above the normal range were found. The results demonstrate that in acute pancreatitis all serum pancreatic enzymes had the same diagnostic sensitivity, however serum lipase determination is the most convenient because of its simplicity and low cost. In chronic pancreatitis serum pancreatic isoamylase and TLI may be useful in detecting severe pancreatic insufficiency. In pancreatic cancer serum pancreatic enzymes lack diagnostic specificity.