Electroporative adjustment of pH in living yeast cells: ratiometric fluorescence pH imaging.

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.

Pseudo real-time method for monitoring of the limiting anisotropy in membranes.

Data acquisition and analysis of the time-resolved fluorescence anisotropy is typically a time consuming process preventing usage of this experimental method for monitoring of time-dependent phenomena. We describe a method for pseudo real-time monitoring of the limiting fluorescence anisotropy r(infinity) allowing to track changes of the membrane order occurring on the time scale of minutes. Principle and performance of the method is demonstrated in the time domain with the time-correlated single photon counting detection. DMPC liposomes stained with 1,6-diphenyl-1,3,5-hexatriene (DPH) have been used to test influence of the diffusion membrane potential on the membrane order during the temperature-induced phase transition in DMPC membranes. It has been found that the transmembrane field of the order of -70 mV increases the phase transition temperature by about 1.5 degrees C-2 degrees C. It is proposed that the full advantage of the method can be utilized with a gated detection, which besides a faster data acquisition brings additional advantage of excitation light suppression. The method can be also used for imaging.

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.

[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.

[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.

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.

[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.

[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.

[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.

[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.

[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.

Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells.

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (tau2 = 2.4 +/- 0.2 ns) to that of the short component (tau1 = 0.4 +/- 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.

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.

[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.

[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.

[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.

[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.

Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae.

Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.

[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.

[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.