One-year results from cryopreserved mitral allograft transplantation into the tricuspid position in a sheep experimental model.

Mitral allografts are still used only exceptionally in the mitral or tricuspid position. The main indication remains infectious endocarditis of atrioventricular valves for its flexibility and low risk of infection. The aim of our study was to evaluate 1-year results of mitral allografts transplantation into the tricuspid position in a sheep model. Mitral allografts were processed, cryopreserved, and transplanted into the tricuspid position anatomically (Group I - 11 animals) or antianatomically (Group II - 8 animals). All survivors (4 from Group I, and 3 from Group II) were checked at 3, 6, and 12 months by echocardiography with the exception of one survivor from Group II (which was examinated only visually). Examination throughout follow-up included for mitral allograft regurgitation and annuli dilatation. At postmortem, the papillary muscles were healed and firmly anchored to the right ventricular wall in all subjects. Transventricular fixation of the papillary muscles with buttressed sutures was proven to be a stable, reproducible, and safe method for anchoring mitral allograft leaflets. There were no significant differences between the two implantation methods. Annulus support of mitral allografts might be very useful in this type of operation and could prevent annular dilatation.

Pt-CeO2 coating of carbon nanotubes grown on anode gas diffusion layer of the polymer electrolyte membrane fuel cell.

The growing of carbon nanotubes on a gas diffusion layer (GDL) was investigated using electron microscopy and photoelectron spectroscopy. The 30 nm thick Pt doped CeO2 layers were deposited by (rf) magnetron sputtering using a CeO2-Pt target on a carbon diffusion layer overgrown by carbon nanotubes. The anode prepared in such a way was tested in the proton exchange membrane fuel cell. Hydrogen/air fuel cell activity measurements normalized to the amount of used Pt revealed high specific power (W mg(-1) Pt). The high activity of this anode with CNT-grown is explained by high specific area of the catalyst, high conductivity of CNT-GDL junction and high activity of platinum present in cationic state Pt2,4+. Very high specific power and low cost together with physical vapor deposition of catalyst makes this anode preparation promising for micro fabrication of fuel cells to power mobile systems.

Platinum-doped CeO2 thin film catalysts prepared by magnetron sputtering.

The interaction of Pt with CeO(2) layers was investigated by using photoelectron spectroscopy. The 30 nm thick Pt doped CeO(2) layers were deposited simultaneously by rf-magnetron sputtering on a Si(001) substrate, multiwall carbon nanotubes (CNTs) supported by a carbon diffusion layer of a polymer membrane fuel cell and on CNTs grown on the silicon wafer by the CVD technique. The synchrotron radiation X-ray photoelectron spectra showed the formation of cerium oxide with completely ionized Pt(2+,4+) species, and with the Pt(2+)/Pt(4+) ratio strongly dependent on the substrate. The TEM and XRD study showed the Pt(2+)/Pt(4+) ratio is dependent on the film structure.

Rats with monosodium glutamate-induced obesity and insulin resistance exhibit low expression of Galpha(i2) G-protein.

In order to test the potential role of inhibitory G-proteins in mechanisms of insulin resistance in adipose tissue of obese animals we determined the content of Galpha(i1) and Galpha(i2) proteins and an extent of protein tyrosine phosphorylation in epididymal fat tissue cell membranes using immunoblot. Monosodium glutamate-induced obese rats displayed adipose tissue hypertrophy, elevated levels of insulin, leptin and slightly elevated serum glucose. We found significantly decreased protein content of Galpha(i2) in adipose tissue plasma membranes of obese rats. This was in accordance with lower protein tyrosine phosphorylation noticed in adipose tissue cell homogenate of glutamate-treated animals. Our results confirm the role of Galpha(i2) in development of insulin resistance by crosstalk between the reduced level of inhibitory G-protein and insulin receptor mediated most likely by activation of phosphotyrosine protein dephosphorylation.

Evaluation of serum and urine clusterin as a potential tumor marker for urinary bladder cancer.

Clusterin is a stress-associated cytoprotective chaperone up-regulated by various apoptotic triggers in many cancers and neurodegenerative diseases. No valid information about serum or urine clusterin concentration in patiens with bladder cancer exists. Aim of our paper was evaluation of the urine and serum clusterin concentrations in individuals with bladder cancer. Blood and urine samples were used from 43 patients with urothelial tumors of the urinary bladder and from 50 patients with benign urological diseases. Blood and urine were collected before cystoscopy. Enzyme-linked immunosorbent assays (ELISA) were performed for clusterin from serum and urine. Serum clusterin was higher in individuals with bladder cancer (means 185,812.5 vs 171,946.5 kU/l, p=0.04). Sensitivity for bladder cancer detection was 73% and specificity 55% (AUC 0.63); efficacy was not sufficient. Urine values of clusterin were higher in individuals with bladder cancer (197.2 vs 67.7, p=0.0007). Sensitivity for bladder cancer detection was 49% and specificity 92% (AUC 0.75, LR+ 6.1, PPV+ 84%); diagnostic efficacy was sufficient. In conclusion, serum and urine clusterin can differ between bladder cancer patients and the control group. Urine clusterin could be the possible laboratory marker of bladder cancer. Further research is warranted to confirm findings in larger studies of various clinical status.

Complex therapy of advanced colorectal carcinoma.

The present paper reports on a complex therapy of 18 patients with primary unresectable advanced carcinoma of the rectum and rectosigmoid. The results of surgery following complete chemoradiotherapy are evaluated. Radical surgery was successful in 15/18 patients. The authors describe a high incidence of postoperative complications and point out a high erudition of an oncosurgeon necessary for such intervention as well as for the indication of a patient to this extensive operation. (Tab. 2, Ref. 18.)

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents.

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

[Personal experience with treatment of posttraumatic urethral distraction defects]. / Nase zkusenosti s lécbou posttraumatických uretrálních distrakcních defektu.

PURPOSE OF THE STUDY: Authors present their experience in the treatment of posttraumatic distraction urethral defect resulting from traumatic rupture of posterior urethra. MATERIAL: The group comprised 19 patients with posttraumatic urethral distraction defect (average age 41 year, range 27-65 years). In 16 of them (84%) resection urehtroplasty was performed and in three (16%) endoscopic internal urethrotomy was applied. The patients were evaluated of 19 to 48 months after surgery. METHOD: Urethroplasty was performed at least three months after the trauma, always under general anesthaesia in lithotomic position, using perinal approach. Dissection of bulbar urethra was followed by dissection and resection of fibrous posttraumatic distraction defect (the original membranous urethra). Prostatic apex and proximal end of lumbar urethra were spatulated and bulboprostatic anastomosis was performed restoring urethral continuity. A catheter was left in urethra for three weeks. In 12 patients it was necessary to separe corpora cavernosa addition and 5 patients required a wedge resection of the lower arch of public bones to allow urethral bridge the defect. Endoscopic internal urehtrotomy was also performed minimally three months after trauma, always on position 12 of the clock face opposite to symphysis with a discision of the whole stenotic part. Subsequently, catheter was inserted in urethra and left in place for four days. RESULTS: Resection urethroplasty as primary surgery was successful in 15 (94%) patients and only 1 patients (6%) required another reconstruction surgery. Endoscopic management was not successful in any patients (100%). Two of them (66%) had to undergo repeatedly a reconstruction surgery, the third one (33%) is regularly dilated. All patients after urethroplasty are under regular circumstances continent, only in two of them (13%) there occurs of urine in case of an extreme increase of abdominal pressure. Erectile function already impaired by the trauma did not worsen by the surgery in 4 patients (25%), in 2 patients (13%) with preoperatively normal erections there developed erectile dysfunction after urethroplasty of which in 1 patient a permanent disorder. The quality of life was in general evaluated by patients as excellent. DISCUSSION: Epicystotomy is a simple procedure ensuring urinary diversion in patients with posterior urethral rupture. However, such management of urethral rupture almost always results in the development posttraumatic distraction defect. Incontinence occurs in our group only in 2 (12%) patients, mainly in non-standard situations (gym, urgency). Night incontinence does not occur in our patients at all. Continence is in our patients ensured by lissosfincter which is fully sufficient. Erectile dysfunction may result from a trauma or a treatment. In our group all patients have a preserved erection prior to trauma and trauma was evident cause of the loss of erection only in 2 (12%) patients who were primarily treated by epicystotomy. In another 2 patients (12%) who were primarily treated after trauma for coincidental urinary bladder rupture it is impossible to state what caused the erectile dysfunction whether a fracture or surgery. In the acute phase during the revision of the rupture of posterior urethra the peroperative risk of the impairment of neurovascular bundles responsible for erection is much higher than in planned surgery. Satisfaction of patients with the treatment is reflected in the evaluation of the postoperative results and the quality of life in general. None of our patients managed by delayed internal urethrotomy was cured. One is regularly dilated, another two underwent urethroplasty. CONCLUSION: The technique of resection of urethral distraction defects with bulboprostatic anastomosis is a suitable way of the treatment of the preceding rupture of posterior urethra without impairement of continence or erection. A prerequisite of good results is a simple urine diversion by epicystostomy during the primary management of the posterior urethral rupture. Delayed endoscopic therapy of the distraction defect will not probably cure the patients but will result in regular dilatations. It may be an alternative treatment in polymorbid or biologically older patients.

Hydrogen bonding effects on the (15)N and (1)H shielding tensors in nucleic acid base pairs.

The results of systematic ab initio calculations of (15)N and (1)H chemical shielding tensors in the GC base pair as a function of hydrogen bond length are presented for the first time. The hydrogen bond length characterized by the distance r(N...N) between purine N1 and pyrimidine N3 was varied between 2.57 and 3.50 A and the chemical shift tensors were calculated by the sum-over-states density functional perturbation theory. It is shown that the hydrogen bond length has a strong effect on the chemical shielding tensor of both imino proton and nitrogen, on their orientation, and, as a consequence, on the relaxation properties of both nuclei. For a nitrogen nucleus not involved in hydrogen bonding, the shielding tensor is nearly axially symmetric and almost collinear with the bond vector. As the length of the hydrogen bond decreases, the least shielding component sigma(11) deflects from the N-H vector and the shielding tensor becomes increasingly asymmetric. The significance of the presented results for the analysis of relaxation data and the efficiency of TROSY effects together with a summary of the relevant shielding parameters are presented and discussed.

Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids.

Triple resonance HCN and HCNCH experiments are reliable methods of establishing sugar-to-base connectivity in the NMR spectra of isotopicaly labeled oligonucleotides. However, with larger molecules the sensitivity of the experiments is drastically reduced due to relaxation processes. Since the polarization transfer between 13C and 15N nuclei relies on rather small heteronuclear coupling constants (11-12 Hz), the long evolution periods (up to 30-40 ms) in the pulse sequences cannot be avoided. Therefore any effort to enhance sensitivity has to concentrate on manipulating the spin system in such a way that the spin-spin relaxation rates would be minimized. In the present paper we analyze the efficiency of the two known approaches of relaxation rate control, namely the use of multiple-quantum coherence (MQ) and of the relaxation interference between chemical shift anisotropy and dipolar relaxation - TROSY. Both theoretical calculations and experimental results suggest that for the sugar moiety (H1'-C1'-N1/9) the MQ approach is clearly preferable. For the base moiety (H6/8-C6/8-N1/9), however, the TROSY shows results superior to the MQ suppression of the dipole-dipole relaxation at moderate magnetic fields (500 MHz) and the sensitivity improvement becomes dramatically more pronounced at very high fields (800 MHz). The pulse schemes of the triple-resonance HCN experiments with sensitivity optimized performance for unambiguous assignments of intra-residual sugar-to-base connectivities combining both approaches are presented.

Base dynamics in a UUCG tetraloop RNA hairpin characterized by 15N spin relaxation: correlations with structure and stability.

Intramolecular dynamics of guanine and uracil bases in a 14-nt RNA hairpin including the extraordinarily stable UUCG tetraloop were studied by 15N spin relaxation experiments that are sensitive to structural fluctuations occurring on a time scale of picoseconds to nanoseconds. The relaxation data were interpreted in the framework of the anisotropic model-free formalism, using assumed values for the chemical shift anisotropies of the 15N spins. The rotational diffusion tensor was determined to be symmetric with an axial ratio of 1.34 +/- 0.12, in agreement with estimates based on the ratio of the principal moments of the inertia tensor. The model-free results indicate that the bases of the G x U pair in the tetraloop are at least as rigid as the interior base pairs in the stem, whereas the 5'-terminal guanine is more flexible. The observed range of order parameters corresponds to base fluctuations of 19-22 degrees about the chi torsion angle. The results reveal dynamical consequences of the unusual structural features in the UUCG tetraloop and offer insights into the configurational entropy of hairpin formation.

Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex.

BACKGROUND: Aminoglycoside antibiotics are known to target ribosomal, retroviral and catalytic RNAs with high affinity and specificity. Recently, in vitro selection experiments have identified RNA aptamers that bind to aminoglycoside antibiotics with nanomolar affinity and stringent specificity, allowing discrimination between closely related family members. There has, to date, been limited structural information on the molecular basis of such saccharide-RNA recognition. RESULTS: We describe a solution-structure determination of the tobramycin-RNA aptamer complex, obtained using NMR and molecular dynamics. The structure gives insight into the molecular features associated with saccharide-RNA recognition. Tobramycin adopts a defined alignment and binds to the RNA major groove centered about a stem-loop junction site. A portion of the bound tobramycin is encapsulated between the floor of the major groove and a looped-out cytosine residue that forms a flap over the binding site in the complex. CONCLUSIONS: The emergence of antibiotic-resistant pathogens and their impact on human health continues to be a major concern in the medical community. Rational modification of existing antibiotics aimed at improving their efficacy requires a molecular view of their receptor-binding sites. We have provided such a molecular view for a member of the aminoglycoside antibiotic family that targets RNA.

[Personal experience with peroperative study of testicular blood flow in laparoscopic surgery of varicocele]. / Nase zkusenosti s peroperacním sledováním prokrvení varlete pri laparoskopické operaci pro varikokélu.

Although the authors did not detect in 27 patients operated since October 1996 laparoscopically for varicocele, although the spermatic artery was ligatured a deteriorated blood supply of the testis, they evaluated the blood flow through the spermatic artery in the rete testis by coloured Doppler mapping during temporary peroperative ligature of the spermatic artery. In 14 patients the occlusion did not cause any changes of the blood supply of the testis, in one patient during occlusion of the artery the Doppler signal in the rete testis disappeared, after release of the ligature the blood flow reappeared in the coloured Doppler mapping. The authors conclude that in the great majority of men the blood supply of the testis is ensured by collateral circulation. If the ligature of the spermatic artery is inevitable they recommend to check during operation the blood supply of the testis in the rete testis by dopplerometry.

[Ultrasonographic changes in prostatic tissue after thermotherapy]. / Zmeny prostatické tkáne po termoterapii v ultrazvukovém obraze.

The authors evaluated in 13 patients the peroperative and postoperative effect of thermotherapy by transrectal ultrasonography. In all patients for a period of 60 minutes by means of a transurethral cooled catheter a temperature of 50 degrees C was applied. In the two-dimensional B picture in all patients a hypoechogenic border round the urethra was observed which gradually became defined as a canal of varying width. Changes in the blood supply of the prostate were not detected by the authors. The functional equivalent of these changes was an improved uroflowmertric finding and IPPS score.

[Urodynamic changes after transureteral microwave thermotherapy of benign prostatic hyperplasia]. / Urodynamické zmeny po transuretrální mikrovlnné termoterapii benigní hyperplazie prostaty.

In 32 patients with benign prostate hyperplasia different urodynamic parameters were investigated before treatment by transurethral microwave thermotherapy and after this treatment. After three months positive changes were recorded as regards the storage as well as micturition phase which did not change during the subsequent six months. Transurethral microwave therapy is a suitable therapeutic method of benign hyperplasia of the prostate in mildly obstructive patients.

[Importance of determination of post-micturition of residual urine in patients with benign prostatic hyperplasia]. / Význam stanovení postmikcní reziduální moci u nemocných s benigní hyperplazií prostaty.

Assessment of the residual post-micturition urine is considered by many urologists an important examination in patients with benign prostate hyperplasia. The residue is found in these patients more frequently than in the healthy population. However, it does not always correlate with the uroflowmetric findings. Following analysis of results of urodynamic studies in 315 selected patients with isolated benign prostate hyperplasia the author concludes that the finding of a significant amount of post-micturition residual urine draws the clinician's attention to impaired micturition but does not define the type of disorder nor its grade. It is of importance only in isolated benign prostate hyperplasia, if higher than 100 ml.

[Endosonography of the ureter--initial experience with a new method]. / Endosonografie mocovodu--první zkusenosti s novou metodou.

The authors had the opportunity to examine four patients, using a new intraureteral sensing unit and to study the basic ultrasonographic picture of the whole ureter. In addition to the ascendent application of the sensing unit they used also the descendent approach during percutaneous lithotripsy and peroperative exploration of the ureter during ureterotomy. The authors assume that the method will prove useful in particular for detection and assessment of the position of renal vessels before endopyelotomy in hydronephrosis.

RNA folding topology and intermolecular contacts in the AMP-RNA aptamer complex.

We report below on the NMR structural characterization of the complex between AMP and a 40-mer RNA aptamer in aqueous solution. Resonance assignments are based on multinuclear multidimensional NMR studies on complexes uniformly 13C, 15N-labeled with either AMP or the RNA aptamer. AMP binds to an internal loop (labeled G7-G8-A9-A10-G11-A12-A13-A14-C15-U16-G17) and bulge (G34 positioned opposite the internal loop) segment in the RNA aptamer, and our NMR study provides insights into features of the RNA folding topology and the molecular recognition events in the AMP binding pocket on the RNA. Specifically, the helical stems are extended by G-G mismatch formation from either direction into the internal loop/bulge segment of the RNA aptamer on complex formation. The internal loop adopts a unique fold with the purine ring of AMP intercalated between A10 and G11 in the complex. The G8-A9-A10-AMP segment adopts certain stacking features in common with a GNRA turn and is closed by the G7.G11 mismatch pair. The purine rings of A12 and G34 (syn) are stacked on each other and participate in stablizing the AMP intercalation site. A large number of intermolecular NOEs have been identified between the AMP ligand and the G8, A10, G11, G17, U18, and G34 residues on the RNA aptamer in the complex. The Watson-Crick edge of the AMP is oriented toward the exocyclic amino group of G8, suggestive of a hydrogen-bonding alignment between G8 and AMP in the complex. The AMP sugar ring is positioned in the minor groove of the rightward helical stem centered about the G17.G34 mismatch and U18.A33 Watson-Crick pairs. The AMP binds to one face of the folded internal loop/bulge segment of the RNA aptamer while the opposite face is capped by a stacked alignment of the A13-A14-C15-U16 segment located toward the 3'-end of the internal loop segment. Globally, the two helical stems of the RNA aptamer are aligned approximately orthogonal to each other with tertiary interactions centered about the internal loop/bulge segment generating the AMP binding site on the RNA.

Molecular recognition in the FMN-RNA aptamer complex.

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.