Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.     

Increase in specific density of levobupivacaine and fentanyl solution ensures lower incidence of inadequate block

The clinical presentation of a subarachnoid block (SAB) is dependent upon the intrathecal spread of local anesthetic (LA). Intrathecal distribution depends on the chemical and physical characteristics of LA, puncture site, technique used, patient anatomical characteristics and hydrodynamic properties of cerebrospinal fluid. We tried to determine whether a combined glucose/LA solution can render a clinically significant difference in sensory block distribution and motor block intensity.This was a controlled, randomized and double blinded study. The surgical procedures were stripping of the great or small saphenous vein and extirpation of remaining varicose veins. The study included 110 patients distributed into two groups: Hyperbaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 10% glucose (Pliva)) vs. Hypobaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 0.9% NaCl (Pliva, Zagreb)) adding to a total volume of 3.5 ml per solution. Spinal puncture was at L3-L4 level. Spinal block distribution was assessed in five minute intervals and intensity of motor block was assessed according to the modified Bromage scale. Pain was assessed with the Visual Analogue Scale. A statistically significant difference in sensory block distribution, motor block intensity and recovery time was established between hyperbaric and hypobaric solutions. By increasing the specific density of anesthetic solution, a higher sensory block, with lesser variability, a diminished influence of Body Mass Index, decreased motor block intensity and faster recovery time may be achieved.     

Voice rehabilitation of Broca's aphasia following total laryngectomy

Total laryngectomy, as a consequence of carcinoma of the larynx, results in loss of speech function. Cerebrovascular stroke is the leading cause of reduced speech production ability, and thereby communication difficulties. The case is presented of a 60-year-old male patient who suffered stroke five years after a total laryngectomy. Speech rehabilitation was hampered due to the depressive state of the patient. Although contraindicated, the secondary voice prosthesis was implanted. Only at that moment the patient showed willingness and motivation for speech rehabilitation. The aim of this presentation is to demonstrate that not all neurological disorders are contraindicated for implantation of voice prostheses.     

Effective conductivity of a suspension of permeabilized cells: a theoretical analysis

During the electroporation cell membrane undergoes structural changes, which increase the membrane conductivity and consequently lead to a change in effective conductivity of a cell suspension. To correlate microscopic membrane changes to macroscopic changes in conductivity of a suspension, we analyzed the effective conductivity theoretically, using two different approaches: numerically, using the finite elements method; and analytically, by using the equivalence principle. We derived the equation, which connects membrane conductivity with effective conductivity of the cell suspension. The changes in effective conductivity were analyzed for different parameters: cell volume fraction, membrane and medium conductivity, critical transmembrane potential, and cell orientation. In our analysis we used a tensor form of the effective conductivity, thus taking into account the anisotropic nature of the cell electropermeabilization and rotation of the cells. To determine the effect of cell rotation, as questioned by some authors, the difference between conductivity of a cell suspension with normally distributed orientations and parallel orientation was also calculated, and determined to be <10%. The presented theory provides a theoretical basis for the analysis of measurements of the effective conductivity during electroporation.     

Application of short monolithic columns for fast purification of plasmid DNA

A simple method for the separation of the four major neutral glycosphingolipids, present in all human tissue, was developed. This gradient normal phase-HPLC method utilises a polyvinyl alcohol bonded stationary phase and an evaporative light-scattering detection (ELSD). Screening pure solvents in a binary gradient elution mode allowed, in a first step, to assess the behaviour of the studied solutes and to select the solvents for further mobile phase optimisation. The proportion of the remaining solvents was defined to reach a maximal resolution. The reduction of the analysis time and the enhancement of the signal were obtained by optimising the gradient slope and the flow-rate. Optimal levels of triethylamine and formic acid (TEA-FA) for the enhancement of the evaporative light scattering detector response were established at 0.1% (v/v). Thus, the optimal conditions for the separation of the four glycosphingolipids was obtained with a gradient elution from a 100% chloroform to a 100% acetone:methanol (90:10 (v/v)) mobile phase at 0.2 ml min-1, using a 10% min-1 gradient slope. Finally, this method was applied to detect the excess of one of the neutral sphingolipids, namely globotriaosylceramide (Gb3) in the urine of patients affected with Fabry disease. A liquid-liquid extraction of the sediments obtained from an aliquot of only ten ml of urine proved sufficient to detect the excess of Gb3 present in both hemizygote and heterozygote patients. In all, the ability of our method to detect abnormal amounts of Gb3 in urinary sediments could allow the diagnosis of weakly symptomatic Fabry patients in large screening programs     

Probability distribution on the parse trees

Paper describes probability distribution on the parse trees of natural language by using Bayesian networks. First parts of the paper describes probabilistic context-free grammar and parse trees. In the second part of the paper, Bayesian network was modelled and joint probability distribution on their vertex. On these theoretical ideas, in the third part, we describe our model tested on Database of grammatical sentences of Croatian language (http:/ /infoz.ffzg.hr / tepes /). At the end was presented a backward procedure and evaluation of our results.     

Diagnostic value of a peroral sucrose permeability test in children with recurrent upper abdominal pain

The access of ingested sucrose into blood and urine indicates the presence of mucosal lesions in the upper gastrointestinal tract. The study involved 42 children, aged 5-15, having recurrent upper abdominal pain and 43 peers with minor extra-abdominal complaints. Sucrose in urine was determined by thin layer chromatography. The test was positive in 27 out of 42 children having recurrent abdominal pain (64.3%) and in none of the control children (chi2 = 37.6, p < 0.0001). When correlated with endoscopic findings it was falsely negative in 12 out of 38 patients with endoscopically verified lesions of the stomach or duodenum and falsely positive in 1 out of 4 without lesions. Sensitivity of the test was 68.4%, specificity 97.9%, positive predictive value 96.3%. The test cannot be used as an alternative to endoscopy, but may serve for screening of candidates for it.     

Measurement of tumor oxygenation using new frequency domain phosphorometers

Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.     

Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells

Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP) via Toll-like receptors TLR2 and TLR4, and stimulate immune and inflammatory responses leading to sterile inflammation and propagation of already existing inflammation. It was found elevated in the blood of patients with chronic obstructive pulmonary disease (COPD), who might suffer occasional bacterial colonizations and infections. We used a monocytic THP-1 cell line as a cellular model of systemic compartment of COPD to assess inflammatory effects of eHsp70 when present alone or together with bacterial products lypopolysaccharide (LPS) and lypoteichoic acid (LTA). THP-1 cells were differentiated into macrophage-like cells and treated with various concentrations of recombinant human Hsp70 protein (rhHsp70), LPS (TLR4 agonist), LTA (TLR2 agonist), and their combinations for 4, 12, 24, and 48 h. Concentrations of IL-1α, IL-6, IL-8, and TNF-α were determined by ELISA. Cell viability was assessed by MTS assay, and mode of cell death by luminometric measurements of caspases-3/7, -8, and -9 activities. rhHsp70 showed cell protecting effect by suppressing caspases-3/7 activation, while LPS provoked cytotoxicity through caspases-8 and -3/7 pathway. Regarding inflammatory processes, rhHsp70 alone induced secretion of IL-1α and IL-8, but had modulatory effects on release of all four cytokines when applied together with LPS or LTA. Combined effect with LPS was mainly synergistic, and with LTA mainly antagonistic, although it was cytokine- and time-dependent. Our results confirmed pro-inflammatory function of extracellular Hsp70, and suggest its possible implication in COPD exacerbations caused by bacterial infection through desensitization or inappropriate activation of TLR2 and TLR4 receptors.