Diffusion membrane potential in liposomes: setting by ion gradients, absolute calibration and monitoring of fast changes by spectral shifts of diS-C3(3) fluorescence maximum.

A novel fluorescent technique for direct assessment of membrane potential was tested on suspensions of large unilamellar vesicles (LUV). The method is based on monitoring shifts in the fluorescence maximum, lambda(max), of the redistribution dye diS-C3(3) caused by dye binding to the LUV membrane. A theory describing the behavior of this dye in LUV suspensions was elaborated and tested. The diffusion potentials across the LUV membrane were adjusted by ion gradients in the absence of valinomycin. When using KCl and choline chloride without valinomycin the potential can be set as high as -70 mV. These potentials exhibited long-term stability and the theory allowed to determine the upper limits of membrane permeabilities for Cl-, choline cations, protons and hydroxyls relative to the K+ permeability. The absolute values of membrane potential set by ion gradients were calibrated using valinomycin. The monitoring of the lambda(max) shift permitted us to show real-time changes in membrane potential, since addition of valinomycin to the LUV was followed by an immediate depolarization. The setting of the potential and the dye re-equilibration after valinomycin addition took place within a second.

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles.

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane. The generated Delta(psi) was stable at least over tens of minutes. An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.

Fluorescence behavior of the pH-sensitive probe carboxy SNARF-1 in suspension of liposomes.

When exposed to the intracellular environment fluorescent probes sensitive to pH exhibit changes of photophysical characteristics as a result of an interaction of the dye molecule with cell constituents such as proteins, lipids or nucleic acids. This effect is reflected in calibration curves different from those found with the same dye in pure buffer solutions. To study an interaction of the probe 5'(and 6')-carboxy-10-dimethylamino-3-hydroxy- spiro[7H-benzo[c]xanthene-7,1'(3H)-isobenzofuran]-3'-one (carboxy SNARF-1) with membrane lipids, we measured its fluorescence in model systems of large unilamellar vesicles (LUV) prepared by extrusion. When the dye was removed from the bulk solution by gel filtration the relative fluorescence intensity of the lipid-bound dye form was enhanced, showing a strong interaction of the dye molecule with LUV membrane lipids. Surprisingly, the dye molecules seem to be bound predominantly to the outer surface of the lipid bilayer. The same situation was found with small unilamellar vesicles prepared by sonication. This effect makes it difficult to use carboxy SNARF-1 for measurements of the internal pH in suspensions of liposomes.

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells.

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.

[Ethylene glycol poisoning]. / Otrava etylenglykolem.

Ethylene glycol is a common substance in various antifreeze preparations. Intoxication is caused by ingestion either as a suicide attempt or by an accident. The clinical course of intoxication is always severe, however, immediate launching of proper treatment can reverse the poor prognosis. Intoxication clinically proceeds in three stages: 1. Depressive CNS disturbances that can lead to coma. 2. Organ manifestations. 3. Acute renal failure. The case report of 46-year-old man admitted after ingestion of ethylene glycol in suicidal attempt summarises the essential principles of therapy--monitoring of vital functions, administration of ethanol solution as an antidote and hemodialysis to remove toxic components.

[Secondary hemophagocytic syndrome in a systemic disease]. / Sekundární hemofagocytující syndrom pri systémovém onemocnení.

20 year old man 2 years treated for the seropositive rheumatoid arthritis was admitted for fever accompanied with jaundice, anemia and leukopenia. The underlying disease has been compensated already for long period of time, before his admission only Prednisone (in the dose of 5 mg daily) and Methotrexate (15 mg once a week) was given. His physical examination of admission was without any significant abnormalities, out of the routine laboratory examination the value of leukocytes count was 2.1 x 10(9)/L, erythrocytes 3.7 x 10(12)/L, hemoglobin 95 g/l, hematocrit 0.29, platelets 156 x 10(9)/L. Since admission to hospital the hepatic enzymes ALT, AST, GMT, ALP were about ten times elevated comparing to normal values, the coagulation examination has shown the decrease of Quick test to 55%. With respect to the permanent leukopenia the bone marrow aspiration was taken with the finding of the increase number the RES elements (18.4%) with the signs of hemophagocytosis. The phagocytic reticulum absorbs blood elements erythrocytes, normoblasts, granulocytes, platelets. According to the literature experience we started the combination of the immunosuppressive treatment consisting of corticosteroids and Cyclosporine. Already the day following the application of the high dose of corticosteroids the fever subsided, icterus went away gradually with the normalization of the liver tests. After 20 days of hospitalisation the patient was discharged in good shape. Now, after 4 months the is stabilized on the follow-up treatment of Prednisone a Cyclosporine.

[Pulmonary edema caused by laryngospasm]. / Laryngospazmem indukovaný plicní otok.

The authors report on a case of non-cardiac pulmonary oedema that developed in a young athletic man at an operating theatre. This unusual condition resulted from a laryngospasm followed by strenuous ventilation retractions. Diagnostic routine, differential diagnosis and treatment are described.

[Poisoning in patients treated during 4 years at the Metabolic Unit of the Second Internal Medicine Clinic of the Charles University Medical School in Prague]. / Intoxikace u nemocných osetrených v prubehu ctyr let na Metabolické jednotce interní kliniky 2. Lékarské fakulty Univerzity Karlovy v Praze.

The authors summarize their experience with intoxicated patients who were treated between 1994 and 1997 at the metabolic unit of the Medical Clinic, Second Medical Faculty Charles University, Prague. The group of intoxicated patients comprises 165 subjects, incl. 76 men and 89 women. 107 intoxications were committed with suicidal intentions, in another 27 cases intoxication was accidental, in 14 toxicomania was involved and 17 patients were chronic alcohol addicts. From the total number 57 patients suffered from serious psychiatric disease of the type of psychosis, but only 22 patients had a psychiatric case-history. In 46 instances suicide was a demonstrative deed or hysterical reaction to partnership conflicts. In this group women predominate over men. The group comprises 14 toxicomaniacs, incl. 9 dependent on hard drugs. The patients were most frequently intoxicated with alcohol which they took either alone or combined with medicaments. 56 of the patients consumed ethyl alcohol. The next most frequently used intoxicating substance is flunitrazepam (Rohypnol), which was taken by 48 subjects. In 113 cases medium severe intoxications were involved, 31 were mild and 21 intoxications were severe.

[The short bowel syndrome]. / Syndrom krátké klicky.

The authors demonstrate on case-histories their experience with short-loop syndrome which develops after extensive resections of the small intestine. The clinical picture of the syndrome is characterized by diarrhoea, steatorrhoea, maldigestion and malabsorption with loss of body weight. Non-surgical treatment of the syndrome copies its different stages. The objective of conservative therapy is above all to preserve the nutritional integrity of the organism and gradual adaptation of the gut which will make eventually a change to oral intake possible. This adaptation takes 1 to 2 years. Last not least, treatment should eliminate the diarrhoea. The authors demonstrate on examples that properly conducted treatment prevented the development of malnutrition, contrary to another case where the sequelae of surgery were underestimated and the patient developed cachexia and pernicious anaemia.

[Realimentation in patients with anorexia nervosa and critical malnutrition--personal experience]. / Realimentace pacientu s mentální anorexií a kritickou malnutricí--nase zkusenosti.

The objective of the work is to find a suitable realimentation procedure for patients with anorexia nervosa and critical malnutrition, whose mortality is round 6%. In a group of 10 patients aged 22.3 +/- 2.33 years during hospitalization for 10.0 +/- 6.11 days a weight increment of 5.0 +/- 1.16 kg was achieved. According to hitherto assembled experience and consistent with data in the literature it is best to start immediately after admission complete parenteral nutrition with a hyperalimentation regime regardless of the amount of food ingested by the patient. Thus within the shortest time maximum weight increments are achieved and the patients can be referred to the care of psychiatrists, which is the only causal treatment. Attempts of realimentation by enteral nutrition or increased oral ingestion in these patients were not successful and led only to a prolonged period of hospitalization.

[Evaluation of nutritional state in hospitalized patients with cerebrovascular stroke]. / Hodnocení stavu výzivy u hospitalizovaných pacientu s cévní mozkovou príhodou.

The cerebrovascular stroke is serious disease with the high mortality and resulting in invalidity. The result of its treatment is not yet encouraging. The stress situation which is characteristic for the acute stage of disease leads to catabolic status. This condition can be worsen if nutritional parameters are not properly monitored and the nutrition is not sufficient. We examined 30 patients admitted to our medical ward with the diagnosis of ischemic cerebrovascular stroke. We have not applied any nutritional intervention. We measured some anthropometric, biochemical and imunological parameters of nutrition at the time of admission to ward and after 14 days of hospitalization. Our results showed that nutritional status has worsen during this period according to the significant deterioration of the followed anthropometric and biochemical parameters. Malnutrition has the detrimental impact on rehabilitation and mobilization of the patients and extends time of hospitalization. Nutritional status should be monitored and corrected since the start of disease, preferably in intensive care unit.

[Reversible long-term tachycardia and hypertension as a symptom of autonomic neuropathy in combination with sensorimotor polyneuropathy in a patient with newly discovered type 1 diabetic mellitus]. / Reverzibilní dlouhodobá tachykardie a hypertenze jako projev autonomní neuropatie v kombinaci se senzorickomotorickou polyneuropatií u nemocné s cerstvým záchytem diabetes mellitus 1. typu.

The case of 22 years old woman admitted with ketoacidotic coma and newly diagnosed insulin dependent diabetes mellitus is described. The signs of mixed sensoromotoric polyneuropathia in this patient have been discovered. After the correction of ketoacidotic hyperglycemic coma the significant tachycardia and hypertension with the abnormalities of diurnal rhythm with necessity of the intensive treatment persisted for the period of the several monthes. These changes we attributed to the significant dysfunction of the autonomic system. In the course of 1 year of good diabetes compensation the above mentionned hemodynamic changes subsided completely. In the same time the signs of mixed polyneuropatia and the incipient retinopathia disappeared. The causes of the described changes are discussed, mainly the importance of reversible microvascular changes.

[Severe sepsis with organ failure in necrotizing fasciitis of the right leg caused by infection with Vibrio vulnificus]. / Tezká sepse s orgánovým selháním pri nekrotizující fasciitide pravého bérce vyvolané infekcí Vibrio vulnificus.

Vibrio vulnificus is a human pathogen which can cause the septicemia and necrosis of the soft tissue, especially fasciitis. The occurrence is most common in the summer, the source of infection can be sea water or the sand on the beaches, however the infection after eating seafood was described as well. The patient with predominant liver dysfunction are more often affected. The clinical course is characterized by severe sepsis, and massive skin lesion. The mortality more than 60% is reported. This case report describes the course of disease in 64 year old patient, who has spent his vacation in Bulgaria. After return he was admitted with severe sepsis with multiorgan failure and despite the intensive therapy and high amputation of the limb which was performed, the patient died.

[Postural trauma and rhabdomyolosis causing acute renal failure]. / Pozicní trauma s rabdomyolýzou a následným akutním renálním selháním.

Rhabdomyolysis (damage of the muscles of various origin) leads to the efflux of the intracellular fluids in the circulation. The common complication of this status is the renal failure. The early diagnosis and the proper treatment makes the fall of renal function reversible. That is why the possibility of the rhabdomyolysis must be consider. The case report describes the development of renal failure in young, previously healthy men, followed by trauma mechanism after drug and alcohol abuse.

[Tissue amino acids in patients with colorectal carcinoma]. / Tkánový aminoacidogram u nemocných s kolorektálním karcinomem.

Different catabolic conditions have their special characteristics of intracellular biochemical changes. The objective of the presented work is to assess the concentration of free amino acids in muscles of patients with colorectal carcinoma. In a group of 17 patients the free amino acid level was assessed in tissues and plasma. Material was collected on operation by biopsy of the rectus abdominis muscle, the concentration of different amino acids was assessed by the HPLCV method with fluorescent detection. For statistical evaluation the T-test was used. From the results ensues that in patients with colorectal cancer in plasma statistically significantly lower taurine, glutamine, valine, tyrosine levels were found, intracellularly significantly reduced levels of taurine, glutamic acid, methionine and ornithine were recorded. Significantly elevated intracellular levels of valine, isoleucine, leucine, tyrosine and phenylalanine were found. Assessment of the tissue aminoacidogram is the first step when attempting to influence the intracellular amino acid concentration by defined dietetic preparations.

[Malnutrition in a patient with short-bowel syndrome]. / Malnutrice nemocného se syndromem krátkého streva.

The short bowel syndrome is a relatively frequent complication of extensive resection and by reducing the absorption capacity the function of the gut is markedly impaired. An important part of postoperative care is therefore to ensure nutrition of these patients. The authors demonstrate on the case-history of a 40-year-old patient after repeated revision of the abdominal cavity on account of adhesions the development of severe malnutrition after jejunotransversoanastomosis. As the small intestine was not resected (concurrent blind loop syndrome), the patient was indicated for surgery. After surgery all complaints disappeared and the nutritional status improved.

[A rare case of necrotizing fasciitis and subsequent sepsis with multiorgan failure caused by Vibrio vulnificus]. / Vzácný prípad nekrotizující fasciitidy a následné sepse s multiorgánovým selháním zpusobený agens Vibrio vulnificus.

The authors report a rare case of necrotizing fasciitis, sepsis caused by a strain of Vibrio vulnificus rare in Czech Republic.