Study of alkaloid berberine and its interaction with the human telomeric i-motif DNA structure.

The alkaloid berberine presents many biological activities related to its potential to bind DNA structures, such as duplex or G-quadruplex. Recently, it has been proposed that berberine may interact with i-motif structures formed from the folding of cytosine-rich sequences. In the present work, the interaction of this alkaloid with the i-motif formed by the human telomere cytosine-rich sequence, as well as with several positive and negative controls, has been studied. Molecular fluorescence and circular dichroism spectroscopies, as well as nuclear magnetic resonance spectrometry and competitive dialysis, have been used with this purpose. The results shown here reveal that the interaction of berberine with this i-motif is weak, mostly electrostatics in nature and takes place with bases not involved in C·C+ base pairs. Moreover, this ligand is not selective for i-motif structures, as binds equally to both, folded structure, and unfolded strand, without producing any stabilization of the i-motif. As a conclusion, the development of analytical methods based on the interaction of fluorescent ligands, such as berberine, with i-motif structures should consider the thermodynamic aspects related with the interaction, as well as the selectivity of the proposed ligands with different DNA structures, including unfolded strands.

Determination of deuterium oxide content in water based on luminescence quenching.

Water molecules (H2O) often reduce luminescence lifetimes of various luminescence probes. The change of lifetime is usually caused by dynamic luminescence quenching induced by O-H oscillators which effectively take away energy from excited molecule. The process can be described by Stern-Volmer equation. We have studied selected luminescence systems where it is possible to detect considerable changes of lifetime in presence/absence of H2O and D2O in this work for analytical purposes. We have tested both, inorganic (Ln3+) and organic compounds using three different instrumentation in order to find the largest change between τH and τD. The Ln3+ containing systems have shown considerable increase/decrease of lifetimes in the presence/absence of D2O (Eu3+: τD/τH = 34.5) whereas organic systems gave significantly lower values of τD/τH (coumarin 123 lifetime ratio, τD/τH = 1.94). The calculated LOD varied from 0.04 mol l-1 (samarium nitrate) to 6.55 mol l-1 (riboflavin).

A study on bioluminescence and photoluminescence in the earthworm Eisenia lucens.

Eisenia lucens is an earthworm living in the organic soil layer of decomposing wood. When irritated, the worm expels coelomic fluid through pores in its body wall, exhibiting blue-green bioluminescence. The mechanism of the bioluminescence, which seems to be different from other bioluminescence systems of terrestrial animals, has been studied in this work. Many lines of evidence indicate that riboflavin stored in coelomycetes plays an important role in this glowing reaction.

In vivo 31P MR spectroscopy of human kidney grafts using the 2D-chemical shift imaging method.

The aim of this work was to evaluate the possibility to use in vivo (31)P magnetic resonance spectroscopy (MRS) for the diagnosis of kidney graft dysfunction after transplantation. We examined 68 kidney grafted patients using a 1.5 T MR scanner. (31)P MRS was performed using the 2D-chemical shift imaging method. The patients were divided into 4 groups: acute rejection episode; acute tubular necrosis; late graft dysfunction; or good renal function. We measured the signal intensities of phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), and α-, ß-, γ-adenosine triphosphate (ATP; with contributions of α- and ß-adenosine diphosphate) and their ratios. Patients with an acute rejection episodes showed a significantly elevated PME/ß-ATP and PDE/ß-ATP, PME/Pi, and PDE/Pi signal ratios compared with the control group. The group with acute tubular necrosis had decreased ratios. Patients with late graft dysfunction revealed only an insignificant decrease in PME/Pi and PDE/Pi ratios. We concluded that (31)P MRS was capable of distinguishing the two main causes of graft dysfunction early after transplantation.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS) study of Huperzine A, a natural anti-Alzheimer's disease product, its derivatization and its detection by highly sensitive laser induced fluorescence (LIF).

Huperzine A, a reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (HupA), was studied using an (MALDI TOF MS) instrument in MALDI mode. The formation of a HupA dimmer in a vacuum was observed and several matrices were found that were able to inhibit its formation. The structures of the neutral and protonated form of the HupA molecule were calculated and optimized using a Hyperchem program. Detection limit using MALDI TOF MS in the model sample was 5.3pg. MALDI TOF MS was also applied to the direct detection of the drug in medical preparations and in human serum. The limit of detection in plasma was 14.2pg with a signal-to-noise ratio of 3:1. However, the sensitivity was not as high as it usually is in MALDI. Therefore, a new method for the derivatization of HupA was developed using fluorescent labelling with rhodamine B isothiocyanate (RBITC). A limit of detection using capillary electrophoresis laser induced fluorescence detection (CE-LIF) equal to 4x10(-9)moll(-1) was reached.

[Nitric oxide (NO) in a model of renal ischemia and cyclosporine toxicity]. / Oxid dusnatý (NO) u modelu ischémie ledvin a cyklosporinové toxicity.

BACKGROUND: The role of nitric oxide (NO) after the cadaveric kidney transplantation has not been fully clarified yet. The aim of our study was to examine benefits of the administration of a NO precursor--L-arginine in the model of renal ischaemia and after the subsequent cyclosporine (CsA) treatment, which simulates the state resulting from the kidney transplantation. METHODS AND RESULTS: 60 male rats of the Wistar strain were exposed to ischaemia for 45 minutes. Then they were divided into six groups: 1. Controls, 2. Rats administered by gastric sonde with 300 mg/kg of L-arginine since the first day after ischaemia, 3. Rats administered in a similar way with 10 mg/kg of cyclosporine A, 4. Group of rats receiving both drugs in the same doses, 5. Rats receiving 10 mg/kg of cyclosporine A since the first day after ischaemia and L-arginine in the dose 300 mg/kg since the seventh day, 6. Group of animals administered with L-arginine and cyclosporin A in the same doses with nonselective blocker of NO synthesis--L-NNA in the dose of 5 mg/kg. We examined renal functions (blood and urine levels of creatinine, urea, Na, K, Cl, osmolality, proteinuria), blood and urine levels of NO metabolites (NO2- and NO3-) in the fourth week after ischaemia. We found that L-arginine administration (when groups 1 and 2 were compared) decreased S-creatinine (< 0.05), it increased U-osmolality (p < 0.01), tubular resorption (p < 0.001), and blood levels of NO metabolites (p < 0.05). Changes in urine levels of NO metabolites (U-NOx/U-Cr) and in proteinuria were not found. In animals with renal ischaemia treated with cyclosporine (comparison of groups 3 and 4), L-arginine administration brought about a decrease of blood creatinine levels (p < 0.05), higher creatinine clearance (p < 0.05) and higher blood levels of NO metabolites (S-NOx; p < 0.01). However, differences in tubular functions, proteinuria and U-NOx/U-Cr were not detected. When L-arginine was added 7 days after the beginning of cyclosporine treatment, no significant difference was found between groups 5 and 3 and differences between groups 5 and 4 were similar to those between groups 4 and 3. Animals of the group 6 were not tested due to high mortality indicating high toxicity of the combination ischaemia + cyclosporine + L-arginine + L-NNA. CONCLUSION: Our study shows the benefits of L-arginine treatment in the renal ischaemia and during cyclosporine administration. The treatment should start immediately after the ischaemic period and at the same time as the cyclosporine administration. L-arginine added later has no positive effect.

[Nitric oxide in patients after cadaveric kidney transplantation]. / Oxid dusnatý u nemocných po transplantaci kadaverózní ledviny.

BACKGROUND: The role of nitric oxide (NO) after cadaveric renal graft transplantation has not been yet fully clarified. The aim of our study was to examine NO production into the urine of patients following cadaveric renal graft transplantation with a normal course and complications (acute rejection and cyclosporin toxicity). METHODS AND RESULTS: Production of stabile NO metabolites (NO2 and NO3) into urine (U-NOx) was examined in recipients of cadaveric renal transplantation. Only patients with standard triple immunosuppressive therapy (cyclosporin, azathioprine, prednisone) were include into the study. Patients receiving other immunosuppressive agents or drugs affecting NO formation (nitrates, ACE inhibitors) were excluded from the study, as were those with infectious or other serious post-transplant complications. Overall, we examined 33 patients (21 men and 12 women), with acute rejection and cyclosporin-induced toxicity in ten each, and a normal course with no complications in 13. The mean age of the patients was 50.96_11.13 years. U-NOx was examined by biochemistry using Griesse reaction every day after transplantation both in a morning urine sample and in a sample from 24-hour collection over the preceding day and calculated to 1 mmol/l of urinary creatinine (U-Cr). The levels of U-NOx/U-Cr in patients with acute rejection over the past 2 days before its development were lower compared with those in patients with a normal course (p_0.05). No difference was found between the groups of patients with cyclosporin-induced toxicity and a normal course. The levels of U-NOx were inversely correlated (p_0.01) to the levels of serum creatinine (S-Cr), but did not correlate with the blood levels of cyclosporin A. CONCLUSIONS: The study demonstrated a decrease in urinary U-NOx production within the past 2 days before renal transplant rejection. The levels of U-NOx in patients with cyclosporin-induced toxicity remain unaltered. U-NOx/U-Cr could possibly become a non-invasive marker of rejection.

[Conversion from Consupren sol. to Consupren S capsules in kidney transplant patients]. / Prevod z Consuprenu sol. na Consupren S kapsle u nemocných po transplantaci ledviny.

In a group of patients after transplantation of the kidney with stabilized graft function treated by Consupren sol. combined with prednisone and azathioprin in 20 patients (group A) Consupren sol. was replaced by Consupren S capsules, in 17 patients (group B) Consupren sol. therapy proceeded without any change. To maintain the cyclosporin blood concentration within the therapeutic range it was necessary after the change of drug form in group A to adjust the dosage of the drug in 12 patients of group A while in group B only in one patient (p<0.01). The mean doses and levels of Cy-A however did not change significantly during the three-month investigation period in the two groups and and the bioequivalence of the two preparations was evident. Conversion from Consupren sol. to Consupren S capsules is not associated with the risk of rejection or undesirable effects. It can be implemented at a ratio of 1:1 or 1: the closest dose divisible by 25 (the smallest capsules are 25 mg) and after conversion a check-up or possible modification of the dose is necessary.

[Progressive multifocal leukoencephalopathy in a female patient after kidney transplantation]. / Progresivní multifokální leukoencefalopatie u nemocné po transplantaci ledviny.

The authors describe the case of a 66-year-old female patient after transplantation of the kidney where a rare complication of immunosuppressive treatment was diagnosed--progressive multifocal leukoencephalopathy. The disease was manifested by gradually developing hemiparesis of the lower extremity five months after transplantation. Examination of the brain by computed tomography and examination of cerebrospinal fluid were normal. Subsequently the neurological finding developed into quadruparesis. Imaging of the brain by magnetic resonance revealed multiple demyelinization foci. In brain biopsy there were typical structures of progressive multifocal leukoencephalopathy and positive hybridization in situ for JC-virus in cell nuclei. Immunosuppressive treatment was restricted and ganciclovirus administration was started. Further progression of the clinical symptomatology ceased, the function of the transplanted kidney remained satisfactory. The patient died 14 months after the transplantation from sepsis. Examination of the post-mortem material revealed positivity of the JC-virus only in the cytoplasm of the affected cells. As effective treatment of infection with JC-virus is not known so far, the possible action of ganciclovirus is only speculative.

[Light chain deposition disease as a cause of renal failure]. / Nemoc z ukládání lehkých retezcu jako prícina renálního selhání.

The objective of the paper is to draw attention to a rare cause of rapidly progressing renal failure which developed in the course of four months as a result of light chain deposition disease. The authors submit two case-histories of the disease assessed by renal biopsy after previous clinical and laboratory suspicion of monoclonal gammapathy. In one patient in the sternal punctate plasmacytoma was diagnosed and in the second case it was not possible to detect any type of monoclonal gammapathy or another possible cause of disease. Renal failure was in both cases irreversible and both patients were enlisted in regular haemodialyzation treatment.

[A single-center controlled clinical study with Sandimmune Neoral after kidney transplantation]. / Monocentrická kontrolovaná klinická studie se Sandimmunem Neoral u nemocných po transplantaci ledviny.

The authors compared in a controlled clinical study two groups of patients after a first renal transplantation treated by triple drug immunosuppressive therapy. In a group of 31 patients the triple combination comprised Sandimmune Neoral. In the control group there were 30 patients who received Sandimmune. No differences were found between the two groups as regards the effectiveness of this treatment and the authors did not confirm a lower incidence of rejections described in patients treated with Sandimmune Neoral. They confirmed, however, a lower interindividual variability of Cy-A levels assessed specifically in patients treated with Sandimmune Neoral.

Potential of 31P magnetic resonance spectroscopy in monitoring the viability of human renal grafts stored in Euro-Collins perfusion solution.

The relative concentrations of inorganic phosphate and phosphomonoesters (PME) of 18 human cadaveric kidneys stored in Euro-Collins perfusion solution were measured by 31P MR spectroscopy. The signals of intracellular inorganic phosphate (Pii) and inorganic phosphate contained in the perfusion solution (Pie) were separated by the deconvolution technique. The ratio of the signal intensities of phosphomonoesters and intracellular inorganic phosphate (PME/Pii) was used as a marker of kidney viability and correlated with kidney function after transplantation. Separation of the Pii and Pie signals in the measured spectra was successful in 72% of kidneys. The results of MR analysis satisfactorily agree in 78% with the post-transplant function of kidneys.

[Dynamic bone scintigraphy in the diagnosis of renal osteopathy]. / Pouzití dynamické kostiní scintigrafie v diagnostice renální osteopatie.

UNLABELLED: A study was undertaken to assess types of RO by a dynamic modification of osteoscintigraphy 53 pts dialyzed for 27-75 mths, 19-52 yrs old were divided, according to histological bone pictures, into 4 groups: I) 11 pts with hyperparathyroidism, II) 12 pts with osteomalacia, III) 11 pts with mixed RO, IV) 11 pts were after parathyroidectomy (PTE) and 13 controls. INVESTIGATIONS: the rate of 99mTc-Sn-HEDP accumulation in the skeleton, skeletal/background radioactivity index both registered for 60 min. (parameters K and P), serum levels of C-PTH, bone isoenzyme of alkaline phosphatase (bALP), acid phosphatase (ACP), free hydroxyproline (S-Hypro). RESULTS: 1. compared to controls: elevations of K and all biochemical parameters, P in groups I and III (p < 0.01 - < 0.001) were found. 2. Group 1 was characterized by the highest values of all parameters compared to groups II and IV (p < 0.01). 3. Linear relationships were found between K and bALP (p < 0.01), P and bALP, ACP, S-Hypro (p < 0.01) in pts of groups I, II, III. 4. PTE in group IV was followed by a decrease in all parameters (p < 0.01) compared to values of the same pts in group I. Aluminum osteopathy was present in 2/3 of cases in group II showing the lowest values of all parameters. CONCLUSION: DBS is a sensitive method for assessing bone turnover rate according to the degree of skeletal uptake of radionuclide. In this manner it is possible to determine both the type of RO and it's further development in repeated measurements.

[Changes in plasma renin and aldosterone after parathyroidectomy in patients with hyperparathyroidism]. / Zmeny plazmatického reninu a aldosteronu po paratyroidektomii u nemocných s hyperparatyrózou.

The plasma renin activity and its changes after parathyroidectomy indicate a preserved internal secretory renal function in dialyzed patients. The PRA values before parathyroidectomy are not unequivocally related to the blood pressure reading. After parathyroidectomy during the initial months the renin and aldosterone plasma levels decline in patients with secondary and primary HPT (p < 0.001), the urinary Na/K quotient rises in primary HPT (p < 0.05) and the systemic blood pressure declines in dialyzed patients with secondary HPT (p < 0.001). The findings suggest relations between the two hormonal systems during hyperparathyroidism and in the early stage after parathyroidectomy. Parathormone probably stimulates renin secretion. After a prolonged time interval following operation the parathormone levels in the blood steam reach normal levels and the same probably applies to intracellular calcium in cells of the iuxtaglomerular apparatus along with PRA.

31P magnetic resonance spectroscopy investigation of skeletal muscle metabolism in uraemic patients.

Using the method of in vivo magnetic resonance spectroscopy we examined 17 patients with moderately advanced chronic renal insufficiency, 21 patients with chronic renal failure treated by haemodialysis, and 15 dialyzed patients with symptomatic renal osteopathy. The ratios of intracellular phosphocreatine and inorganic phosphate concentrations of these subjects measured at rest were compared with those found in healthy controls. While we noted significantly lower (p < 0.01) ratio values in all patients, subjects with osteopathy showed a lower value than dialyzed patients free of bone disease. Haemodialysis improved the result of examination in 7 patients. The results can be summarized as follows: (1) patients with altered renal function have significantly impaired energy metabolism of skeletal muscle, and (2) the disorder is more severe in patients with renal osteopathy than in those free of it.

Lack of stimulating effect on thyrotropic and lactotropic secretion during prolonged calcitriol administration.

The stimulating effect of calcitriol, administered in the dose of 3 micrograms/day for four days, on the thyrotropic and lactotropic secretion has been proved in earlier studies in healthy humans. The present study was undertaken to confirm or eliminate the stimulatory action of the drug using increasing dosages of 0.75-1.50 micrograms/day for one month. In spite of the fact, that the biologically efficient levels of calcitriol were attained (confirmed indirectly by the rise of urinary calcium excretion, p less than 0.01), the stimulating effect of calcitriol on the basal and TRH induced thyrotropic and lactotropic secretion has not been manifested. In conclusion, the clinical importance of stimulating effect of calcitriol on the thyrotropic and lactotropic secretion is neglected, if only commonly accepted therapeutic dosages up to 1.50 micrograms/day are used.