Synthesis and properties of macrolones characterized by two ether bonds in the linker

In this paper synthesis of macrolones 1–18 starting from azithromycin is reported. Two key steps in the construction of the linker between macrolide and quinolone moiety, are formation of central ether bond by alkylation of unactivated OH group, and formation of terminal C–C bond at 6-position of the quinolone unit. Due to the difficulty in formation of these two bonds the study of alternative synthetic methodologies and optimization of the conditions for the selected routes was required. Formation of C-4″-O-ether bond was completed by modified Michael addition, whereas O-alkylation via diazonium cation proved to be the most effective in formation of the central allylic or propargylic ether bond. Comparison of Heck and Sonogashira reaction revealed the former as preferred route to the C–C bond formation at C(6) position of the quinolone unit. Most of the target compounds exhibited highly favorable antibacterial activity against common respiratory pathogens, without significant cytotoxicity profile when tested in vitro on eukaryotic cell lines.     

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.     

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.     

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.     

Stability of Minimum Essential Medium functionality despite l-glutamine decomposition

l-Glutamine (l-Gln) instability in liquid media is a well-known fact. Also, negative effect of ammonia, one of the l-Gln degradation products, on viability of many cell cultures and on replication of different viruses has been described. However, negative effects of ammonia have been reported in doses excessively exceeding those that could be generated in regularly used liquid culture media due to spontaneous l-Gln breakdown (below 2 mM). Traditional virus vaccine production processes have been established and registered involving l-Gln containing media use. Eventual culture media replacement in the regular production process belongs to the major regulative changes that require substantial financial expenses. The aim of this study was to evaluate the effect of storage of Minimum Essential Media with Hanks salts on their relevant biological functions during virus vaccine production process in relation to l-Gln decrease. Our results show a cell type dependent effect of spontaneous l-Gln degradation during medium storage. They also suggest that for cell cultures used in measles, mumps, and rubella virus production the media retain their functionality in respect to cell viability or virus growth over a certain time window despite l-Gln degradation.     

The Zagreb Skull Collection—The unique identified collection of human skulls from fetuses to centenarians

Skull anatomy and development have been extensively studied due to their significance in evolutionary biology, forensic anthropology, and clinical medicine. Bone collections are an indispensable resource for conducting such anthropological and anatomical studies. However, worldwide there are only few skull collections containing specimens covering the entire fetal and postnatal period. Herein we describe the Zagreb Skull Collection, an identified collection comprising more than 1100 skulls and skull bone sets from the early fetal period to centenarians. The Zagreb Skull Collection consists of two main parts: the unique Collection of Skull Bones containing 386 sets of separated skull bones from the early fetal period to adulthood and the Collection of Skulls containing 742 skulls (age range 4–101 years). The collection was the core source for numerous anatomical studies on the development, postnatal changes, and anatomical variations of the skull. However, the Zagreb Skull Collection is still an underexploited resource for anthropological, forensic, and anatomical studies with translatability to contemporary clinical practice.     

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.     

Nutrient-enhanced productivity in the northern Gulf of Mexico: past,present and future

Nutrient over-enrichment in many areas around the world is having pervasive ecological effects on coastal ecosystems. These effects include reduced dissolved oxygen in aquatic systems and subsequent impacts on living resources. The largest zone of oxygen-depleted coastal waters in the United States, and the entire western Atlantic Ocean, is found in the northern Gulf of Mexico on the Louisiana/Texas continental shelf influenced by the freshwater discharge and nutrient load of the Mississippi River system. The mid-summer bottom areal extent of hypoxic waters (<2 mg l^–1 O₂) in 1985–1992 averaged 8000 to 9000 km² but increased to up to 16000 to 20700 km² in 1993–2001. The Mississippi River system is the dominant source of fresh water and nutrients to the northern Gulf of Mexico. Mississippi River nutrient concentrations and loading to the adjacent continental shelf have changed in the last half of the 20th century. The average annual nitrate concentration doubled, and the mean silicate concentration was reduced by 50%. There is no doubt that the average concentration and flux of nitrogen (per unit volume discharge) increased from the 1950s to 1980s, especially in the spring. There is considerable evidence that nutrient-enhanced primary production in the northern Gulf of Mexico is causally related to the oxygen depletion in the lower water column. Evidence from long-term data sets and the sedimentary record demonstrate that historic increases in riverine dissolved inorganic nitrogen concentration and loads over the last 50 years are highly correlated with indicators of increased productivity in the overlying water column, i.e. eutrophication of the continental shelf waters, and subsequent worsening of oxygen stress in the bottom waters. Evidence associates increased coastal ocean productivity and worsening oxygen depletion with changes in landscape use and nutrient management that resulted in nutrient enrichment of receiving waters. A steady-state model, calibrated to different observed summer conditions, was used to assess the response of the system to reductions in nutrient inputs. A reduction in surface layer chlorophyll and an increase in lower layer dissolved oxygen resulted from a reduction of either nitrogen or phosphorus loading, with the response being greater for nitrogen reductions.