Quantification of the APE2 gene expression level in Candida albicans clinical isolates from patients with diagnosed fungal infections.

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1% agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity.

Tetrazole derivatives bearing benzodiazepine moiety-synthesis and action mode against virulence of Candida albicans.

Tetrazole and benzodiazepine derivatives attract widespread attention due to their remarkable pharmaceutical potential. 5-(2-bromophenyl)-7-fluoro-1-[3-(5-(4-chlorophenyl)-2H-tetrazol-2-yl)propyl]-1,3-dihydro-2H-1,4-benzodiazepin-2-one (6c) and 5-(2-bromophenyl)-7-fluoro-1-[3-(5-(2-chlorophenyl)-2H-tetrazol-2-yl)propyl]-1,3-dihydro-2H-1,4-benzodiazepin-2-one (6d) were selected using the microdilution approach and because of their preferential fungicidal activity toward C. albicans. 6c or 6d altered the hyphal morphology, chitin deposition and membrane permeation in planktonic and sessile cells. The tetrazoles caused PS translocation and the accidental dependent permeabilization (ADP) of sessile cells; 6d showed CNB1-dependent action in candidiasis. The exosome Rh123-rich vesicles extruded out of the membrane as an element of the self-defense detoxification strategy when the treatment, especially with 6d, was conducted. The tetrazoles affected the C. albicans biofilm's viability, and Rh123 sequestrates led to a heterogenous sub-cellular localization. While 6c-dependent sequestration into membranes or sub-cellular organelles was noted, Rh123 cellular loading in control cells and distributed within cells was observed (partitioning into subcellular membranes and organelles in apoptotic cells). In conclusion, we propose new agents aimed at Candida virulence factors triggering ACD without toxicity against eukaryotic cells.

Discrimination between the enzymatic activities of Candida albicans pleomorphic forms determined using the api® ZYM test.

Enzymatic activity profiles for two morphotypes of 37 Candida albicans clinical isolates were compared. Yeast and hyphal forms were grown using yeast extract-peptone-glucose broth or undiluted human serum, respectively. Both morphotypes were documented under scanning electron microscopy. The api(®) ZYM (BioMérieux, France) test was used to evaluate the enzymatic activity profiles for particular pleomorphic forms. None of the examined enzymatic activities showed good agreement (kappa, κ > 0.80) for the two morphotypes of the tested strains. Only leucine arylamidase activity in blastoconidia and hyphae of 35 out of 37 strains appeared to be in significant agreement (κ = 0.770). This phenomenon should be explored further for clinical benefits. For morphotypes of all tested strains, activity profiles of 11 hydrolytic enzymes demonstrated weak agreement (κ = 0.044-0.197). Moreover, satisfactory (κ = 0.218-0.348) and moderate agreement (κ = 0.413-0.479) were noted for enzymatic activity values of five and two enzymes, respectively. The distinct differences in activity profiles of hydrolytic enzymes between hyphae and blastoconidia is suggested to be related to the specific roles of these two morphotypes in particular steps of pathogenesis. Moreover, both morphotypes should be examined by strain biotyping methods. Beta-N-hexosaminidase (HexNAcase) activity assessed by the api(®) ZYM test and on CHROMagar Candida(®) medium (Becton Dickinson, USA) is also discussed.

Does the type of exercise affect tryptophan catabolism in horses?

Tryptophan (Trp) is an essential amino acid which metabolises via the kynurenine pathway to generate a number of bioactive substances referred to as kynurenines. Among those are 3-hydroxykynurenine (3-HKyn) and quinolinic acid, which are neurotoxic, as well as kynurenic acid (Kyna) and xanthurenic acid (XA), which, similarly to nicotinamide (NAm), show neuroprotective and anti-depressive effects. Routine exercise is known to modulate Trp metabolism in skeletal muscle and is thus believed to reduce the risk of depressive states in humans and laboratory animals. Analogously, it was hypothesised that exercise can influence Trp metabolism in horses as well. The aim of this study was to evaluate the influence of two different types of exercise on Trp metabolism in horses of the same breed. A total of 32 purebred Arabian horses were involved in the study. The 22 three-year-old racehorses were subjected to short-time intense exercise. Ten other horses were made to perform endurance competitions at a distance of 80 km. Blood samples were collected at rest and following the end of the exercise period. Plasma concentrations of Trp, kynurenine (Kyn), Kyna, 3-HKyn, XA and NAm were determined using Ultra-High Performance Liquid Chromatography-Electrospray Ionisation-Tandem Mass Spectrometry. Short-time intense exercise led to an increase in plasma concentrations of Kyn, Kyna and XA. The endurance effort induced an increase in Kyna and a decrease in Trp and NAm levels. Both types of exercise, short-time intensive exercise and endurance exercise induced an increase in Trp metabolites, especially Kyna, and did not induce an increase in Trp level. Thus, from a pathophysiological perspective of the kynurenine pathway's influence on mental state, both types of exercise induced beneficial effects in horses.

Mechanisms of cancer metastasis: involvement of cancer stem cells?

The leading cause of death from cancer is tumor expansion, which usually leads to dissemination and metastasis of malignant cells. Accumulating evidence suggests growing tumors contain some very rare primitive cells that are mobile and thus endowed with metastatic potential. If these cells survive radio/chemotherapy, they are responsible for tumor re-growth after treatment. In this review, we discuss the origin of these cells, which: 1) are true cancer stem cells (CSCs) that initiate tumor growth and are subsequently responsible for metastatic dissemination; or 2) are derived from transformed tumor cells by the epithelial mesenchymal transition phenomenon. We also address major molecular mechanisms involved in trafficking of these cells during metastasis, paying special attention to the underappreciated side effects of radio/chemotherapy that may induce pro-metastatic environments in various organs. Overall, we envision that the process of pathological metastasis of cancer cells reflects a physiological property of normal SCs for their ability to migrate, as seen during embryogenesis. Finally, we discovered highly migratory, very small embryonic-like SCs that are deposited during development in adult tissues. As we hypothesize, these cells could: 1) give rise to some primitive types of tumors; and 2) may have a direct role in cancer expansion by being involved in tumor angiogenesis and formation of tumor stroma.

Triclocarban Disrupts the Epigenetic Status of Neuronal Cells and Induces AHR/CAR-Mediated Apoptosis.

Triclocarban is a phenyl ether that has recently been classified as a contaminant of emerging concern. Evidence shows that triclocarban is present in human tissues, but little is known about the impact of triclocarban on the nervous system, particularly at early developmental stages. This study demonstrated that triclocarban that was used at environmentally relevant concentrations induced apoptosis in mouse embryonic neurons, inhibited sumoylation, and changed the epigenetic status, as evidenced by impaired activities of HDAC, sirtuins, and DNMT, global DNA hypomethylation, and alterations of methylation levels of bax, bcl2, Ahr, and Car genes. The use of selective antagonists and specific siRNAs, which was followed by the co-localization of aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR) in mouse neurons, points to the involvement of AHR and CAR in triclocarban-induced neurotoxicity. A 24-h treatment with triclocarban enhanced protein levels of the receptors which was paralleled by Car hypomethylation and Ahr hypermethylation. Car hypomethylation is in line with global DNA hypomethylation and explains the increased mRNA and protein levels of CAR in response to triclocarban. Ahr hypermethylation could reflect reduced Ahr mRNA expression and corresponds to lowered protein levels after 3- and 6-h exposures to triclocarban that is likely related to proteasomal degradation of activated AHR. We hypothesize that the triclocarban-induced apoptosis in mouse neurons and the disruption of epigenetic status involve both AHR- and CAR-mediated effects, which may substantiate a fetal basis of the adult onset of neurological diseases; however, the expression of the receptors is regulated in different ways.

Influence of physical effort on aortic stiffness in young male athletes.

OBJECTIVE: The progression of cardiovascular disease is blunted by regular exercise training as a common non-pharmacological treatment. Recent findings have confirmed that central aortic pressure is more strongly related to cardiovascular events than brachial blood pressure. The aim of the study was to evaluate the influence of a single bout of significant physical effort on central aortic pressure and pulse wave velocity in young male athletes. PATIENTS AND METHODS: The study group consisted of 16 healthy male athletes undergoing regular endurance training. The subjects of the study (21.6 ± 2.85 years of age) underwent a submaximal exercise test consisting of cycling for 30 minutes. Aortic pulse wave velocity (PWV) and derivatives (augmentation index set to 75 heart beats, AIx75; pulse pressure amplification, PPA), ejection duration (ED), subendocardial viability ratio (SEVR) and central blood pressure were examined before and after the exercise test Blood pressure and pulse waveform were evaluated in the supine position after a 15-minute rest by means of the oscillometric method the oscillometric method. RESULTS: Comparing the rest condition to the period immediately following the exercise test, athletes showed lower central than peripheral systolic blood pressure both before (129 ± 11 mmHg and 112 ± 8 mmHg, respectively, p < 0.001) and after (130 ± 10 mmHg to 112 ± 8 mmHg, respectively, p < 0.001) the test. They also showed a decrease of ED from 339.7 ± 44.4 ms to 326.9 ± 41.4 ms (p < 0.02) and an increase of PPA from 136.2 ± 5.4% to 140.3 ± 5.0% (p < 0.02), whereas PWV, AIx75 and SEVR changed insignificantly. CONCLUSIONS: We confirm that PPA is sensitive to an instant change in aortic elasticity. Furthermore, the hemodynamic response to a single physical effort composed of shorter ejection time and increased relative elasticity of the aorta prevents impairment of oxygen supply to the heart musculature.

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells.

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.

The effect of some antibiotic-resistance-conferring plasmids on the removal of the heat-aggregated proteins from Escherichia coli cells.

We found that the presence of plasmids expressing tetracycline resistance or chloramphenicol resistance genes, but not those expressing ampicillin resistance or kanamycin resistance genes, in Escherichia coli led to the retardation of the process of removal of the heat-aggregated proteins (i.e. the S fraction) from the bacterial cells. The presence of chloramphenicol acetyltransferase in the S fraction is demonstrated. Moreover, we observed that the expression of T7 RNA polymerase gene had an influence on S fraction removal. These results suggest that high level production of some heterologous proteins which are accumulated in the cytoplasm, but not proteins exported through the cell membranes, may cause overloading of the S fraction and delay in the removal of heat-aggregated proteins from bacterial cells.

Diagnostic x-ray examinations in Poland in 1986--frequency and type.

This paper presents the results of a survey concerning x-ray diagnostic examinations in Poland as the main source of radiation risk to the general population. The methods of data acquisition and processing are described. In Poland, 21,400,000 patients (572 examinations per 1,000 inhabitants) are annually subjected to diagnostic x-ray examinations. The age and gender structure of examined patients is discussed extensively. Special attention is paid to the share of children and youth in the total number of examined patients. The obtained results are compared with the numerical data for other European countries.

Evaluation of doses to patients in X-ray diagnostics in Poland.

The study includes a detailed analysis of patients' exposure to ionising radiation resulting from diagnostic radiology in Poland. Data concerning the number and types of examinations were collected by wide-range questionnaire surveys conducted in 1986 and 1995. As the result, the number of examinations per 1000 inhabitants was found to be 572 in 1986 and 715 in 1995. Most of the examinations were the basic, conventional ones (mostly chest and spine radiography). A great part of this study includes detailed description of the methodology of evaluating doses received by patients from X-ray procedures. The theoretical Monte Carlo simulation with the original author's computer code was used for this purpose. As the result, the doses to patients in six age groups (< 1, 1-4, 5-9, 10-14, 15-19 and the adults) were estimated. In simulation process the examined persons were represented by the appropriate mathematical human phantoms. The doses from particular examinations performed in exposure conditions routinely used in Polish radiology departments are presented. The mean effective doses for adult patients from typical examinations are: 0.11 mSv from chest, 3.0 mSv from thoracic spine, and 4.3 mSv from L-S spine radiography. Significantly higher doses were received by patients undergoing fluoroscopy procedures: 23 mSv from barium enema and 14 mSv from the stomach examination. In conclusion, the estimated exposure levels in Poland are as follows: the average effective dose per examination: (1.4 +/- 0.6) mSv in 1986, (1.2 +/- 0.5) mSv in 1995, the average effective dose per capita: (0.8 +/- 0.3) mSv in 1986 and 1995.

An update of the frequency and type of diagnostic x-ray examinations in Poland.

This paper presents the results of a survey concerning x-ray diagnostic examinations in Poland, as the main source of radiation risk to the general population. The methods of data collection and processing were similar to that used for the previous national survey (1986). In 1995, 27,600,000 diagnostic x-ray examinations were performed in Poland (6,200,000 more than in 1986). The number of examinations per 1,000 inhabitants increased from 572 to 715 during those nine years. The age and gender structure of examined patients is discussed extensively, the numbers for 1986 and 1995 are compared. Special attention is paid to the proportion of children and adolescents in the total number of patients examined. The obtained results are also compared with the numerical data concerning other countries.

Evaluation of patient exposure in computerised tomogram in Poland.

The increasing number of computerised tomography (CT) procedures performed in Poland in recent years has resulted in a growing contribution of these examinations to the whole exposure of the population to ionising radiation from medical sources. (The number of CT examinations in Poland was 170,000 in 1995 and 460,000 in 1999.) An evaluation is presented of doses to patients in CT examinations performed with different types of CT unit. To evaluate the exposure to patients dose linear product (DLP) was measured using a NOMEX dosemeter with a pencil chamber (PTW, Frieburg) and the cylindrical PMMA phantoms 'head' and 'body'. CTDI values were evaluated according to current methodology as described in European Guidelines (EUR 16262). The measurements were performed for seven types of CT unit made by different companies. The CTDI values were also compared to reference levels recommended by IAEA. In conclusion it was found that the value of collective effective dose (2200 man.Sv), has increased in Poland nearly 4 times in comparison to 1995, whereas the number of CT examinations increased nearly 3 times in this period. For most of the 'controlled' CT scanners the values of CTDI in head procedures are near to or higher than the IAEA Reference Level (50 mGy); this can result from the protocols, which are chosen without a dose analysis.

[Quality assessment of radiological diagnostic examinations from the perspective of radiological protection]. / Ocena jakosci diagnostycznych badan rentgenowskich z punktu widzenia ochrony radiologicznej.

The paper presents current recommendations on radiological protection concerning medical exposure, especially during X-ray diagnostic examinations. Major factors responsible for the level of radiological risk for patients undergoing X-ray examinations are also discussed. (In Poland about 28 million X-ray examinations are performed annually.) A thorough evaluation of 18 X-ray units used in diagnostic laboratories in the Lódz district is presented. It was found that only 6 of 18 X-ray units controlled, satisfy best the evaluation criteria laid down according to the recommendations of the International Atomic Energy Agency (IAEA). The significance of satisfying individual criteria for the achievement of high quality radiological examinations was highlighted.

[Safer radiologic examinations]. / Bezpieczniejsze badania rentgenowskie.

The paper presents a short survey of reasons why high doses are used in diagnostic X-ray examinations and methods for their reduction. Special attention is paid to the use of cassettes with rare-earth intensifying screens. The results of dose measurements made for several combinations of cassette-film are also presented. Patients can receive the lowest doses if it would be possible to use DuPont cassettes with FOTON films.

[Analysis of diagnostic X-Ray units in Poland in view of patients' exposure to ionizing radiation]. / Analiza diagnostycznych aparatów rtg w Polsce pod katem narazenia pacjentów na promieniowanie jonizujace.

An analysis of diagnostic X-ray units in Poland is presented. The air-kerma on the X-ray table was measured for X-ray parameters used routinely during lumbo-sacral spine radiography in a standard patient. The measurements were performed using TLD made of lithium-fluoride. The dose received by patients in different X-ray departments is sometimes higher than two orders of magnitude from 0.14 mGy to 72.11 mGy for the same examination. The dose can be significantly reduced if the cassettes with amplyfying foils, made from rare earth components, are used.

[Radioisotopic diagnostic examination as a factor responsible for the Polish population exposure to ionizing radiation]. / Diagnostyczne badania radioizotopowe jako czynnik narazenia Polskiej populacji na promieniowanie jonizujace (raport wstepny).

For the general population, in vivo radioisotopic radiological examinations are the second, after x-ray diagnostics, artificial source of ionizing radiation used for medical purposes. The 1981 survey was the first review of the radioisotopic examinations in Poland and revealed that 80,000 examinations are performed annually (2 examinations per 1,000 population on average). It was also found that 131 I was the most common isotope used during thyroid examinations very frequently performed at that time. According to the 1996 data published by the National Consultant on Nuclear Medicine, "In Poland there are 36 centers of nuclear medicine within the network of public health care services. Every year about 130,000 radioisotopic examinations are performed." In order to estimate real exposure of the Polish population resulting from radioisotopic diagnostic examinations it is necessary to gather detailed data on the age structure of patients, as well as on the type and activity of radiopharmaceutical be administered. The paper presents the preliminary results of the follow-up study on radioisotopic examinations in Poland carried out by the Department of Radiological Protection, Nofer Institute of Medicine, Lódz. The data have been collected according to the adopted schedule, taking account of the organ (system) examined, the type of administered radiopharmaceutical, the kind and activity of radioactive isotopes used to mark radiopharmaceutics, age and sex of examined patient. By the end of December 2000 the data on radioisotopic examinations performed in 12 centers of nuclear medicine (Warsaw--7; Kielce--2; Gdansk, Lódz and Zgierz--one center in each city) had been collected. Thus far almost 40,000 radioisotopic examinations have been registered. According to the above mentioned procedure it was possible to collect data on all examinations performed in 1998. In addition, general numbers of examinations performed in the centers visited in 1999 were registered; the 2000 data are now being completed. It was finally established that the thyroid and musculoskeletal examinations constitute about 50% of the total number of examinations, which is in agreement with the data applying to the countries classified as those with the first level of health care. However, while an average effective dose per examination in those countries accounts for about 4 mSv, in the Polish centers the analogues indicator, estimated on the basis of the results collected thus far, is about 9 mSv, of this number, over 7 mSv is contributed by thyroid examinations with the use of iodine (131I). The obtained result is a strong stimulus to supplement and complete the data already gathered and to carry out a thorough analysis of radioisotopic examination in view of patients' exposure. Such an analysis would provide the subject matter arguments for the optimization of radiological protection in nuclear medicine in Poland.

[Personnel exposure during interventional radiologic procedures]. / Narazenie personelu podczas zabiegów radiologii interwencyjnej.

Intervention radiology, known also as intravascular surgery, is a new medical specialisation that develops very rapidly. Radiological procedures performed under fluoroscopy include: dilatation of stenosed vessels, recanalization or vascular embolization and angioanastomosis. Although these procedures have been initiated by radiologists, the majority of them are performed now by physicians who are specialised in medical disciplines other than radiology (cardiologists, vascular surgeons, gastroenterologists, etc.). All these specialists are always aware of the fact that during radiological procedures, both the personnel and the patients are at risk of ionizing radiation. For that reason monitoring of the exposure in this occupational group is of particular importance. Bearing in mind that members of surgical teams are often in direct contact with x-ray tube, it is assumed that routine individual dosimetry of staff occupationally exposed to x-rays do not provide adequate assessment of the exposure risk. This paper describes measurements carried out among operating surgeons who perform the following procedures: cardiological interventions (percutaneous transluminal coronary angioplasty (PTCA) and bypass with preceding coronarography); neuroradiology (aneurysm embolization); and intravascular surgery within abdominal cavity (TIPS, nephrostomy). Dosimetric assessment was carried out in operating surgeons who are exposed mostly among the members of surgical teams as they have to be in direct contact with radiation sources. A comprehensive assessment of exposure included the following measurements: equivalent dose for the hands (measured by a specially designed finger dosimeter); equivalent dose for the trunk protected by a lead apron (a dosimeter placed under apron); and equivalent dose for the neck (a dosimeter placed on the upper, external edge of apron). In addition, a dose product and the surface of primary beam were measured (Diamentor dosimeter, PTW, Frieburg) which allowed to define the correlation between the entrance air kerma, measured with thermoluminscence dosimeters, and the amount of primary radiation emitted during the monitored procedure. In all, the surgical teams were monitored during 42 intervention procedures. The results of the study revealed that an operating surgeon is most exposed. The values of an annual effective dose and an annual equivalent dose for the hands and eyes, estimated for individual procedures, were as follows: (a) cardiological angioplastic procedures: effective dose--25 mSv, equivalent dose for the hands--438 mSv, equivalent dose for the eyes--265 mSv; (b) intravascular angioplastic procedures within the abdominal cavity and neuroradiological procedures: effective dose--4 mSv, equivalent dose for the hands--360 mSv, equivalent dose for the eyes--41 mSv. It should be stressed that the aforesaid maximum doses do not exceed relevant standard annual limits binding in Poland.

[Sources of x-rays in school]. / Zródla promieniowania rentgenowskiego w szkole.

Exposure of teachers of physics to x-radiation emitted by instruments which are used for demonstrating rarefied gas discharges during physics lessons at secondary schools is discussed. The measurements performed provide an explicit evidence that an effective, anual exposure dose under the most unfavourable conditions does not exceed admissible levels according to Polish regulations pertaining to persons non-occupationally exposed to ionizing radiation.

[Practical methods used in the system of controlling of x-ray apparatus]. / Praktyczne metody stosowane w systemie kontroli jakosci aparatów rentgenowskich.

An x-ray apparatus like any other device becomes worn out. In order to avoid x-ray examinations of patients performed using a worn out apparatus a periodical control of such appliances should be carried out. This work presents a methodology based on simple quality tests for a standard x-ray apparatus. These tests measure parameters of x-ray tubes and beams of radiation. A positive feature of these tests is that they replace very expensive specialistic measuring equipment.