Strangulation ileus caused by malposition of the ureter.

Ileus is one of the most severe diagnoses of the group of acute abdomen events. Some patients with certain forms of bowel obstruction respond well to conservative therapy. However, the diagnosis of strangulation ileus can be delayed in some situations, resulting in high morbidity and mortality in such cases. Our paper presents the case of an 81-year-old female patient who developed an ileus shortly after ureteral stent placement due to obstructive hydroureteronephrosis. The strangulation of the small intestine was caused by an atypical position of the right ureter in the abdominal cavity.

[Risk Factors for the Development of Distal Tibiofibular Synostosis after Unstable Ankle Fractures]. / Rizikové faktory pro vznik distální tibiofibulární synostózy po luxacních zlomeninách hlezna.

PURPOSE OF THE STUDY The study analyses a cohort of patients with surgically treated ankle fractures who developed complete distal tibiofibular synostoses. It focuses on their occurrence and association with the extent of tibiotalar dislocation of the ankle joint on the trauma X-ray and its relation to the choice of surgery. MATERIAL AND METHODS The cohort of a total of 824 patients with type B and C fractures according to Weber classification was followed up for 9 years. The cohort consisted of 403 (48.9%) men and 421 (51.1%) women. The exclusion criteria included associated talus fractures, calcaneus fractures and fractures of the other bones of the foot. The studied data were obtained retrospectively from medical documentation and by evaluation of trauma X-rays and X-rays obtained during the postoperative checks. The ankle fractures were classified based on the Weber classification and the basic epidemiologic data (age and gender), type of fracture and extent of tibiotalar dislocation of ankle fractures on the trauma X-ray were evaluated. Posttraumatic ankle dislocation was divided into tibiotalar dislocation > 10 mm, tibiotalar dislocation < 10 mm and the group with regular ankle joint. When evaluating the treatment method, the cohort was divided into three groups: Group 1 with one-stage osteosynthesis, Group 2 with temporary K-wire transfixation or external fixation and subsequent secondary conversion to internal osteosynthesis, and Group 3 with definitive transfixation or external fixation of the ankle. The results were statistically evaluated using the Pearson s chi-square test, or the Fisher s exact test for low frequencies. A multivariant logistic regression model was created to identify statistically significant factors contributing to the development of synostosis. The results with the p-value < 0.05 were considered statistically significant. RESULTS In the whole cohort, the synostosis of distal tibiofibular joint was observed in a total of 131 (15.9%) patients. In men it was in 85 (21.1%) cases and in women in 46 (10.9%) cases, which was statistically significant (p < 0.0001). There was a statistically significant difference (p = 0.0020) between the mean age in the group of patients with synostosis (54.4 years) and the mean age in the group of patients without synostosis (49.1 years). Complete distal tibiofibular synostoses were found in 78 (12.7%) fractures classified as type B according to the Weber classification and in 53 (25.5%) type C fractures. When taking into account the gender, synostoses occurred more frequently in men in both types of fractures classified based on the Weber classification, only in type C fractures no statistical significance was established (p = 0.3026). Various size of posttraumatic tibiotalar dislocation was present in both types of fractures. The group with less severe type B ankle fractures showed a statistically significant dominance of synostosis development in cases with large tibiotalar dislocation of more than 10 mm (p<0.0001). In the group with type C fractures different results were obtained. The highest frequency of cases with synostosis was reported in the group with dislocation smaller than 10 mm (p = 0.0698). In the entire cohort, 615 (74.6%) one-stage osteosyntheses were performed and synostoses developed in 77 (12.5%) cases. In 165 (20.0%) patients, transfixation with K-wires or external fixation with subsequent conversion to secondary osteosynthesis were used and synostoses were identified in 50 (30.3%) cases (p < 0.0001). The open fractures showed an insignificantly smaller number of synostoses than the closed fractures (p = 0.5902). DISCUSSION Posttraumatic distal tibiofibular synostoses have varied morphology. A number of studies confirmed that they do not affect much the functional status of the ankle, even despite their extensive finding in the area of syndesmosis is evident on the Xray. Etiologically, a certain role in their development is reported to be played by posttraumatic hematoma in case of damage to deep soft and bony structures of the ankle. CONCLUSIONS A higher occurrence of synostoses was observed in male population, older age patients and also in type C fractures according to the Weber classification. Larger tibiotalar dislocation showed statistical significance in the development of synostoses in type B fractures according to the Weber classification, whereas in type C fractures it was not the main factor contributing to the development of synostosis. In cases where one-staged osteosynthesis was performed, the occurrence of synostoses was statistically significantly lower than in secondary osteosynthesis after temporary stabilisation. Key words: ankle fracture, distal tibiofibular synostosis, ankle joint dislocation, Weber classification, acute surgery, delayed surgery.

[Comparison of Clinical Outcomes of Displaced Diaphyseal Clavicle Fractures]. / Porovnání klinických výsledku lécení dislokovaných zlomenin diafýzy klícní kosti.

PURPOSE OF THE STUDY The aim of this retrospective study was to assess the subjective evaluation of treatment by patients with respect to their return to work and recreational sport following the fracture of clavicle diaphysis with posttraumatic shortening of 1.5 cm, or more, treated non-operatively and surgically. MATERIAL AND METHODS Our group of patients consisted of 51 patients (14 females, 37 males) aged between 18 and 89 years (average age 46 years). We concentrated on the parameters of sex, age, side of injury, extent of posttraumatic shortening, method of treatment, return to work or recreational sport, DASH score at one year post non-operative or surgical treatment. Patients who sustained a pathological fracture, fractures of the clavicle combined with an injury of the acromioclavicular joint or simultaneous fracture of the humerus or the ribs were excluded from the study. Patients with open fractures or re-fractures were excluded as well. The indication for treatment selection was based on pre-operative discussion of the doctor with the patient and the Informed Consent was signed. The patient was informed about different treatment options. A shorter period of fixation of the arm and post-operative physiotherapy was mentioned in connection with surgical treatment as well as potential surgical complications. A statistical analysis comparing the data in both groups was conducted using the Fisher exact test. The p-value of 0.05 or less was considered as statistically significant. RESULTS The right side was affected 26 times, the left side 25 times. The shortening ranged from 1.5 to 3.7 cm. 24 patients (8 females, 16 males) aged 21 to 89 years (average 54 years) were treated non-operatively. 27 patients (6 females, 21 males) aged 18 to 74 years (average 38 years) underwent surgery. The difference in sex distribution in both groups was not statistically significant (p = 0.5311). According to the Robinson classification, there were 17 patients with type 2A2 fractures, of whom 8 underwent surgery and 9 were treated non-operatively, 19 patients with type 2B1 fractures, of whom 9 underwent surgery and 10 were treated non-operatively, and 15 patients with type 2B2, of whom 10 underwent surgery and 5 were treated non-operatively. The surgically treated patients prevailed in type 2B2 only, but this difference was not statistically significant (p = 0.2350). In the non-operatively treated group, 23 out of 24 patients returned to pre-injury activities in 3 months on average. Ten patients (48%) reported reaching the same function as on the other side. In the DASH score evaluation, 11 patients reached the value of 0-3.3, five patients 3.4-10, six patients 10.1-30.0 and two reached the score of more than 30. In the evaluation of capacity to work, 15 out of 24 patients were able to work, 11 of them without any restrictions or difficulties. In the evaluation of the sport and playing musical instrument module, 9 out of 24 patients did not engage in sports activities or do not play any musical instruments. In the surgically treated group, 26 out of 27 patients returned to pre-injury activities within 6 weeks. 19 (70%) patients reported reaching the same function as on the other side. In the DASH score evaluation, 19 patients reached the value of 0-3.3, two patients 3.4-10, 5 patients 10.1-30.0 and one patient with nonunion 72.5. Comparison of the average values of the DASH score demonstrated slightly better results achieved by surgical treatment (9.03 vs 6.77). When assessing the work module, 24 out of 27 patients returned to work, 20 of them without any restrictions or difficulties. Out of 27 patients, 4 patients were no longer able to engage in sports activities or to play a musical instrument. Of the 23 remaining patients, 18 did not have any problems, 5 suffered from minimal problems. The group of patients treated non-operatively included one case of non-union and the same applies to the surgically treated group. In 3 patients the removal of hardware was performed, 3 patients underwent revision of the surgical wound because of infection. DISCUSSION The recommendation of the weight-bearing of the upper extremity was similar in both groups, 12 weeks post injury/surgery on average. It is clear that sooner return to work or sports activities in the surgically treated group was preferred by younger patients who expected quicker recovery. Younger patients were less patient and more eager to return to work and sports, while the older patients, on the other hand, were more cautious about possible complications of surgery. CONCLUSIONS The results of our study did not identify any correlation between the clavicle shortening and the indication for surgical treatment. Surgical treatment was preferred by younger patients, more frequently by males. The rationale was supported by the perspective of sooner return to work and favourite sports activities. Their decision was not affected by the known risks of surgical treatment. Evaluation of the DASH score at one year after injury/surgery showed similar results. A higher incidence of complications in the surgically treated group did not lead to negative evaluation of the selected treatment modality by the highly motivated group of patients either. Key words: fractures of the clavicle diaphysis, non-operative treatment, surgical treatment, return to work, return to sports activities, functional results at 1 year.

[Outcomes and Complications of Surgical Treatment for LSS at 1-Year Follow-up - Prospective Study]. / Výsledky a komplikace po operacním resení LSS v horizontu jednoho roku ­ prospektivní studie.

PURPOSE OF THE STUDY Lumbar spinal stenosis (LSS) is a serious and fairly frequent disorder with prevalence increasing with age which often results in a disability. The surgical procedures are often very risky due to the greater age of patients with severe stenosis and their comorbidities. The study aimed to assess the functional outcomes and complications in patients who underwent surgical treatment for LSS at one year postoperatively and to identify the differences in the functional outcomes, if any, in dependence on the number of operated segments. MATERIAL AND METHODS It was a prospective study which included 33 patients who underwent surgery at the authors department from 1 November 2015 to 1 October 2016 for LSS, the mean age of patients was 69.5 (43-83) years. The surgery was indicated based on the clinical examination, radiography and MR imaging and consisted in posterior decompression with or without stabilisation and fusion. Pre- and post-operatively, a neurological examination and evaluation of patient difficulties were performed by: VAS - particularly for low back pain (VAS-LB) and lower limb pain (VAS-LL), Oswestry Disability Index (ODI) and Swiss Spinal Stenosis Questionnaire (SSSQ). The evaluation was done at a half year and one year after the surgery. The authors made comparisons in dependence on the number of treated segments (1-2, 3-5 and Th-S stabilisation). RESULTS A significant improvement of all the followed-up parameters was reported at one year postoperatively. At one year after the surgery, the VAS-LB score showed a decrease by 2.39 (p = 0.001), the VAS-LL by 3.26 (p < 0.001), while a decrease by 2 or more points is considered clinically significant. When evaluating the SSSQ questionnaire, a decrease in subjective difficulties (SSSQ-S) was by 0.89 (p < 0.001), in physical function (SSSQ-F) by 0.87 (p < 0.001). In this questionnaire, the satisfaction rate with the surgery was 2 - i.e. somewhat satisfied. Altogether 76% of patients were very or somewhat satisfied, no one was very dissatisfied. When evaluating the ODI, an improvement by 20.6% (p < 0.001) was reported. The authors identified neither any significant differences in the outcomes at a one-year follow-up in dependence on the length of stabilisation, nor any significant differences between the six-months and one-year follow-up. Intraoperative complications occurred in 18%, early postoperative complications in 6% of patients. The ASD developed in 9% within one year. DISCUSSION The authors confirmed a significant improvement of the followed-up parameters (VAS-LB, VAS-LL, SSSQ, ODI) which corresponds with recent literature. The decrease in VAS and ODI in the authors study is more marked than the outcomes stated in literature. The authors outcomes clearly show that there is no correlation with the length of stabilisation and the number of decompressed segments. Conversely, the rate of complications was higher in this group than the rates stated in literature, but majority of complications had no consequences for the patients. CONCLUSIONS In the group of patients with a one-year follow-up the authors confirmed that surgical procedures will result in reduced subjective difficulties of patients, reduced pain and improvement of physical function. A significant improvement of all the followed-up parameters was reported. The authors did not confirm the correlation between the postoperative improvement and the number of treated segments. Key words:lumbar spinal stenosis, quality of life, post-operative outcomes, complications.

[Injury of Medial Structures in Ankle Fractures - Epidemiological Aspects and Outcomes of Treatment]. / Poranení mediálních struktur u zlomenin hlezna ­ epidemiologické aspekty a hodnocení výsledku lécby.

PURPOSE OF THE STUDY Ankle fractures are characterised by a high variability of damage to bone and ligament structures which leads to diverse clinical conditions. This study aims to analyse a group of patients with surgically treated ankle fractures, with a focus on evaluating the outcomes of treatment of ankle joint medial structure injuries (medial malleolar fracture, ligament lesions). MATERIAL AND METHODS The analysed group included 186 patients (102 men and 84 women), in whom an ankle fracture surgery was performed in 2015 and 2016. The outcomes of the treatment were evaluated in 111 patients with type B and type C fractures, who underwent a follow-up examination at one year after the surgery consisting in subjective and objective assessment of the condition and an ankle radiograph. The obtained outcomes were processed using the techniques of descriptive statistics and subsequently evaluated through the Pearson´s chi-square test at 5% significance level, or the Fisher´s exact test for low frequencies. RESULTS The mean age of patients in the group was 48.6 years, while it was lower in men than in women (42.8 years compared to 53.9 years). According to Weber classification, 1 % of fractures were classified as a type A fracture, 68 % as a type B, 27 % as a type C. The group of isolated medial malleolar fractures represented 4 % of cases. The medial side of the ankle joint more frequently suffered a ligament lesion (56 %) than a medial malleolar fracture (44 %). The mean age of the patients with a medial malleolar fracture was 51.9 years, whereas the mean age of the patients with a ligament lesion on the medial side of the ankle was 44.2 years. When evaluating the outcomes using the OMA score at one year postoperatively, a statistically significant difference was found (p = 0.002) between the patients with a medial malleolar fracture (OMA 79.9) and the patients with a ligament lesion at the medial side of the ankle joint (OMA 91.2). DISCUSSION The aim of the study was to add more information on medial structures of ankle joint that are of major importance for ensuring stability of ankle fractures. In agreement with the literature, when managing the ankle fractures with an injury suffered on the medial side there is obviously a more uniform approach in cases with medial malleolar fractures. The situation is different in case of the deltoid ligament lesion, when historically there is a certain level of non-uniformity in indications for revision surgeries and treatment of the injured ligament structures. In our group, in the case of ligament lesion on the medial side of the ankle joint an emphasis is put on the fluoroscopy control of the symmetry of tibiotalar joint space before the beginning of the surgery and also after the fibular fracture stabilisation. The revision surgery was indicated in cases where asymmetry of ankle fork was found. The patients considered the treatment outcome better in cases with a ligament lesion than in cases with a medial malleolar fracture. CONCLUSIONS Proper treatment of medial structures of the ankle joint is important for ensuring the stability of ankle fractures. The patients with type B fractures reported better results at one year postoperatively compared to the patients with type C fracture according to the Weber classification. A statistically significantly better results after the ankle fracture surgery were achieved in patients with the presence of a medial ligament lesion compared to the patients with a medial malleolar fracture. Key words:ankle fracture, injury of medial structures, epidemiology, outcomes of treatment.

Beta-hydroxybutyrate in milk as screening test for subclinical ketosis in dairy cows.

Ketosis is a very frequent metabolic disease in dairy cows, resulting in lower milk production, impaired fertility and increased frequency of other diseases. The course of the disease is often subclinical, so early detection is very important. The aim of the study was to investigate the relation between the concentration of beta-hydroxybutyrate in blood and milk and to determine the cut-off value in milk for detection of subclinical ketosis. The study included 94 cows, which were in the first third of lactation. Beta-hydroxybutyrate (BHB) concentrations were measured in blood and milk serum using a biochemical analyser. The average concentration of BHB in the blood serum samples was 1.14 mmol/L while in the milk it was about ten times lower at 0.117 mmol/L. A statistically significant positive correlation between the concentration of BHB in blood and milk (r=0.705, p<0.001) was found. In cows with BHB in blood below 2.0 mmol/L a stronger correlation between blood and milk BHB was established (r=0.658, p<0.001) than in cows with blood BHB above 2.0 mmol/L (r=-0.292, p=0.206). Therefore, BHB in milk is a very suitable indicator in the diagnosis of subclinical ketosis as there is a good correlation between BHB in the blood and milk of cows with subclinical ketosis. The cut-off concentration of BHB in milk set at ≥0.080 mmol/L (AUC=0.91±0.03; p<0.001) is a significant indicator for subclinical ketosis in dairy cows. The sensitivity of the test was 94% and specificity 74%. Beta-hydroxybutyrate in milk is a good indicator of subclinical ketosis in dairy cows and can be measured accurately with a biochemical analyser.

Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells.

Research on brown adipose tissue and its hallmark protein, mitochondrial uncoupling protein UCP1, has been conducted for half a century and has been traditionally studied in the Institute of Physiology (AS CR, Prague), likewise UCP2 residing in multiple tissues for the last two decades. Our group has significantly contributed to the elucidation of UCP uncoupling mechanism, fully dependent on free fatty acids (FFAs) within the inner mitochondrial membrane. Now we review UCP2 physiological roles emphasizing its roles in pancreatic beta-cells, such as antioxidant role, possible tuning of redox homeostasis (consequently UCP2 participation in redox regulations), and fine regulation of glucose-stimulated insulin secretion (GSIS). For example, NADPH has been firmly established as being a modulator of GSIS and since UCP2 may influence redox homeostasis, it likely affects NADPH levels. We also point out the role of phospholipase iPLA2 isoform gamma in providing FFAs for the UCP2 antioxidant function. Such initiation of mild uncoupling hypothetically precedes lipotoxicity in pancreatic beta-cells until it reaches the pathological threshold, after which the antioxidant role of UCP2 can be no more cell-protective, for example due to oxidative stress-accumulated mutations in mtDNA. These mechanisms, together with impaired autocrine insulin function belong to important causes of Type 2 diabetes etiology.

Optical trapping of microalgae at 735-1064 nm: photodamage assessment.

Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.

Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling.

Homeostasis of reactive oxygen species (ROS) in cardiomyocytes is critical for elucidation of normal heart physiology and pathology. Mitochondrial phospholipases A2 (mt-PLA2) have been previously suggested to be activated by ROS. Therefore, we have attempted to elucidate physiological role of such activation. We have found that function of a specific i-isoform of mitochondrial phospholipase A2 (mt-iPLA2) is activated by tert-butylhydroperoxide in isolated rat heart mitochondria. Isoform specificity was judged from the inhibition by bromoenol lactone (BEL), a specific iPLA2 inhibitor. Concomitant uncoupling has been caused by free fatty acids, since it was inhibited by bovine serum albumin. The uncoupling was manifested as a respiration burst accompanied by a slight decrease in mitochondrial inner membrane potential. Since this uncoupling was sensitive to carboxyatractyloside and purine nucleotide di- and tri-phosphates, we conclude that it originated from the onset of fatty acid cycling mediated by the adenine nucleotide translocase (major contribution) and mitochondrial uncoupling protein(s) (minor contribution), respectively. Such a mild uncoupling may provide a feedback downregulation of oxidative stress, since it can further attenuate mitochondrial production of ROS. In conclusion, ROS-induced function of cardiac mt-iPLA2 may stand on a pro-survival side of ischemia-reperfusion injury.

Synthetic study on cystinyl peptides using solution and solid phase methodology: human IgG1 hinge region.

Synthetic study on cystinyl peptides using solution and solid phase methodology was carried out with the central hinge region of immunoglobulin IgG1. In the solid phase synthesis of hexadecapeptide 1c, the time necessary for the formation of disulfide bonds between linear precursors was shortened four times by the action of pure oxygen in buffered solution, in comparison with air oxidation. The product was thus obtained devoid of impurities from side reactions. In the preparation of the shortened bis-cystinyl analogs 2k and 3d of the natural hexadecapeptide 1c, both the classical and polyethylene glycol (PEG6000) solution methods were utilized using a disulfide synthon (Boc-Cys-OPfp)2 to obtain peptide chains in a natural parallel alignment. In the PEG6000 strategy, lysine as a linker on both sides of the polymer was attached to enhance the loading capacity. The leucine residue, instead of proline one, was introduced to the carboxy terminus to facilitate a specific enzymatic cleavage of the peptides from PEG6000 by thermolysine.

Molecular dynamics study of the effect of the gamma-Abu insert on the conformational behavior of the glycopeptide dendrimers based on the oligolysine scaffold in N, N'-dimethylformamide.

Glycodendrimers bearing Tn (alpha-D-GalNAc-(1 --> O)-Ser/Thr), an identified tumor-associated carbohydrate antigen, hold promise in the post-surgery treatment of a variety of tumors such as metastatic breast cancer. We used molecular dynamics (MD) techniques to examine structural differences taking place during synthesis of two classes of tetravalent Multiple Antigen Glycopeptides (MAG) that differ only by the gamma-Abu insert in the structure of the oligolysine core. Each of the selected intermediates of the synthesis was modeled, subjected to the 2 ns run in N,N'-dimethylformamide (DMF) and geometrically characterized. We characterized: a) distances of free, or extended termini from the anchor, b) interatomic distances between free or substituted N termini, c) radius of gyration and d) spatial distribution of molecular density. A detailed conformational analysis of 16 glycodendrimers shows the distinct behavior of the inserted vs. non-inserted constructs already during the first steps of the modeled synthesis. It suggests that the character as well as the length of the insert has a major impact on the spatial characteristics and behavior of the dendritic molecules. The inserts can, in principle, increase a tendency of dendrimers to establish a high-density core, which is similar to the effect of a higher generation.

Human and murine humoral immune recognition of multiple peptides from Schistosoma mansoni glyceraldehyde 3-P dehydrogenase is associated with resistance to Schistosomiasis.

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) is a target of cellular and humoral immune responses of Brazilian and Egyptian subjects putatively resistant to reinfection with S. mansoni. In an aim to develop a safe, stable and effective vaccine based on this promising molecule, six peptides derived from its primary sequence were selected based on the lowest homology to human G3PDH. The synthetic peptides were tested by ELISA against plasma of humans putatively susceptible or resistant to reinfection with S. mansoni or S. haematobium following chemotherapeutic cure of previous infection. Repeat experiments indicated that the six peptides bear human B-cell epitopes that bind immunoglobulin (Ig)M, IgG1 and IgG3 antibodies. Resistance to reinfection appeared to be significantly associated with humoral immune responses to multiple peptides. This contention was supported by studies in the murine model, whereby we examined the B cell immune responses of Swiss and inbred BALB/c and C57BL/6 mice immunized with recombinant SG3PDH (rSG3PDH) to the six SG3PDH-derived peptides. The serum antibodies of rSG3PDH-immunized Swiss mice were directed to only one of the six peptides tested by ELISA. Antibodies from rSG3PDH-immunized C57BL/6 and BALB/c mice bound, respectively, to four and six out of six peptides. In contrast to Swiss mice, immunization of C57BL/6 and BALB/c mice with rSG3PDH induced protection against challenge cercariae which reached the level of significance (P < 0.05) for BALB/c mice. The data together indicate that host recognition of multiple peptides of a candidate vaccine antigen is necessary for the expression of its ability to contribute to protective immunity against Schistosomiasis.

Whole cell biosensors--electrochemical and optical approaches to ecotoxicity testing.

Two different approaches to the interrogation of prokaryotic and eukaryotic cells have been explored, with the objective of developing biosensor based rapid ecotoxicity test protocols for use in a wide range of applications. Prokaryotic cells and some eukaryotic cell types lend themselves to interrogation by mediated amperometry, a technique that allows metabolic activity of the cell to be monitored by accessing cellular redox events with a chemical mediator. Reduction of the mediator by the cell is followed by re-oxidation at an electrode surface poised at a fixed potential. The resulting current flow is proportional to the metabolic status of the cell. A commercial ecotoxicity test, CellSense, employing such whole cell biosensors has now been developed. An alternative approach to interrogation of vertebrate cells has been the use of luminescent reporter genes to determine changes in the metabolic status of the cells following environmental challenge. Several clones have been established of epithelial cells from the bluegill sunfish (BF-2), transfected with the pCIneoLuc plasmid, that express luciferase constitutively. Protocols using these transformed cells are being developed to provide an alternative to the standard neutral red retention test.

Application of capillary isotachophoresis for fruit juice authentication.

A method using capillary isotachophoresis (ITP) was developed and applied for the determination of the anionic profile of orange juices with the aim to obtain some useful information on the authenticity or adulteration of imported and native beverage products. An EA 100 electrophoretic analyser (Villa-LABECO, Slovak Republic) was used for capillary isotachophoretic determination of anions in tested samples. More systems of leading and terminating electrolytes were used. Detection conductivity and UV detection at 254 nm were used. Sample injection volume was 30 microl. These systems allow one to determinate inorganic anions, organic acids and some additives--adulterants in anionic forms in orange juices. By capillary isotachophoretic determination the lengths or areas of characteristic zones were established and compared to authentic orange juices of different species and origin and with RSK reference values (Code of Practice). Special emphasis was placed on D-isocitric acid ITP determination as a reliable fruit juice authentication marker. The presented multicomponent analysis of orange juice authenticity according to ITP anionic profiles obtained by capillary isotachophoresis presents an alternative information source necessary for deciding about authenticity of the products.

Human tumour-associated cell adhesion protein MN/CA IX: identification of M75 epitope and of the region mediating cell adhesion.

MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design.

Determination of glucosinolates using their alkaline degradation and reaction with ferricyanide.

Glucosinolates, a group of naturally occurring thioglucosides, are significant factors impairing the nutritional quality of rapeseed and postextraction rapeseed meal, restricting its use as high-quality protein animal feed. Currently, the European Community standards and Canola definition are being brought in line recommending cultivation and marketing of rapeseed with a glucosinolate content below 18 micromol of total glucosinolates per gram of seeds. Furthermore, some glucosinolates are of increasing interest in Brassica vegetables due to their proven cancer-preventing activities. A novel approach to the analysis of total glucosinolates is reported in this paper based on their alkaline degradation and subsequent reaction of released 1-thioglucose with ferricyanide. The reaction was followed spectrophotometrically using sinigrin and glucotropeaolin as model glucosinolates. The applicability of the method was demonstrated using rapeseed extracts after reducing the interfering effect of phenolics by their adsorption onto polyvinylpolypyrrolidone. Good agreement with official ISO methods was shown.

Peptide and glycopeptide dendrimers. Part II.

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP-SOCs), glycodendrimers and template-assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed.

Stimulation of haemopoiesis and protection of mice against radiation injury by synthetic analogues of muramyldipeptide incorporated in liposomes.

Protection from undesirable effects of radiotherapy or chemotherapy, primarily from myelosuppression, remains still a crucial problem to be studied. Attention has been therefore paid to various immunomodulatory agents that through the monocyte/macrophage system induced production of cytokines, which can induce and operate restoration of haemopoiesis and thus act radioprotectively. Some synthetic analogues of MDP free of undesirable side-effects, were synthesized in the Czech Republic. Lipophilic beta-D-GlcNstearoyl-(1- > 4)-norMurNAc-L-Abu-D-isoGln (DDD-St) was designed to be easily entrapped into liposomes and this liposomal DDD-St protected efficiently mice against irradiation, when administered i.p., i.v. or s.c. 24 h prior to lethal irradiation (survival rate in the range of 30-80% compared with 0% in control). Especially the subcutaneous application of liposomal DDD-St was very efficient. The parameters characteristic of recovery of haemopoiesis in bone marrow on day 10 after 6.5 Gy irradiation were significantly improved in comparison with the controls. Very high radioprotective effect of s.c. administered liposomal DDD-St can be explained (together with induction of haemopoiesis) by an effective and long-lasting activation of nonspecific immunity, which is able to withhold an onset of septicemia in early days after irradiation. In conclusion, the liposomal DDD-St should be therapeutically beneficial in moderating the haemopoietic damage, which is an undesirable effect of radiotherapy or chemotherapy.

Design, synthesis and characterization of bradykinin antagonists via cyclization of the modified backbone.

With the aim of synthesizing cyclic antagonists of the nonapeptide hormone bradykinin with minimal side chain modification, we performed backbone to backbone and backbone to side chain cyclization. To probe and compare different strategies for this new kind of cyclization, the branched peptide bonds were formed by both reductive alkylation on the solid phase and by using preformed building units. Lactam bridges between the modified amide groups were formed by the use of the phenylalanine derivatives N(CH2COOH)Phe and N(CH2CH2NH2)Phe. The best results in the formation of the N-alkylamide bond were obtained with the coupling reagent PyBrop. The coupling rate was monitored by estimation of the N-terminal Fmoc-group. The cyclization was performed on the solid support. Unexpected difficulties resulted from the instability of the N-alkylamide bond under strong acidic conditions, as used for deprotection and for removal from the resin. We synthesized peptides with backbone to backbone cyclization between positions 2 and 5, as well as backbone to side chain cyclizations between positions 0 and 5, and between 2 and 6. The relatively high biological activities of some of the cyclic analogues support the supposed receptor-bound conformation of bradykinin antagonists with a beta-turn in the N-terminal sequence.