Novel glycoside of vanillyl alcohol, 4-hydroxy-3-methoxybenzyl-α-d-glucopyranoside: study of enzymatic synthesis, in vitro digestion and antioxidant activity

Novel glucoside of physiological active vanillyl alcohol was synthesized for the first time using maltase from Saccharomyces cerevisiae as catalyst, and established its structure as 4-hydroxy-3-methoxybenzyl-α-D: -glucopyranoside. The key reaction factors for this transglucosylation reaction were optimized using response surface methodology and the highest yield so far in maltase catalyzed transglucosylation reaction was obtained. It was found out that optimum temperature of reaction was 37 °C, optimal maltose concentration was 60% (w/v), optimal pH was 6.6, and optimal concentration of vanillyl alcohol was 158 mM. Under these conditions, yield of glucoside was 90 mM with no by product formation. It was shown that this compound posses good antioxidant activity as well as stability in gastrointestinal tract. It was demonstrated that it is hydrolyzed on brush border membrane of enterocytes, so it can serve in protecting gastrointestinal system from oxidation, as well as source of anticonvulsive drug after the hydrolysis of glucoside on brush border membrane of small intestine.     

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.     

Structural dependencies of protein backbone 2JNC' couplings

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.     

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4-91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6-4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 +/- 7.3 and 39.6 +/- 6.2% of CM-MSC pairs, 21.9 +/- 5.0 and 13.6 +/- 1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.     

The role of fat dormouse (Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.     

Effect of storage conditions on virulence of Fusarium avenaceum and Alternaria alternata on apple fruits

To overcome difficulty in phytopathogenic fungi control during storage of apple fruits, the effect of different storage conditions on the occurrence and development of Fusarium avenaceum and Alternaria alternata infections on apple cultivar “Cripps Pink” was investigated during and after storage. Inhibitory effects of wild oregano essential oil on apple fruit rots caused by F. avenaceum and A. alternata were also tested as possible rot control measure. Artificially inoculated apple fruits were kept in cold storage with normal (NA) and controlled (CA) atmosphere for 95 days and at room temperature only. The obtained results indicated that different storage conditions significantly affect necrosis development on apple fruits caused by F. avenaceum and A. alternata after storage, as well as during shelf life.     

Gelatinous invertebrate zooplankton of the South Adriatic: species composition and vertical distribution

The species composition and vertical distribution of gelatinousinvertebrate zooplankton were investigated from April 1993 toJune 1994 in the 0–1000 m water column at a deep-sea stationin the northern part of the South Adriatic Pit. Fifty-sevenspecies were identified: 11 hydromedusae, 13 calycophores, 3ctenophores, 3 heteropods, 10 pteropods, 8 polychaetes and 9chaetognaths. The pteropod Desmopterus papilio and the heteropodProtatlanta mediterranea were recorded for the first time inthe Adriatic Sea. Data from this study differed from those ofprevious investigations in the South Adriatic as regards thenumerically dominant polychaete and pteropod species. All investigatedgroups generally were more abundant in the upper 100 m and decreasedwith depth. Different vertical distributions of life stageswere observed for those species that occupy a wide depth range:Persa incolorata, Solmissus albescens, Limacina inflata, Cymbuliaperoni, Pelagobia longicirrata, Sagitta lyra and Sagitta decipiens.     

Mutations in the Sepiapterin Reductase Gene Cause a Novel Tetrahydrobiopterin-Dependent Monoamine-Neurotransmitter Deficiency without Hyperphenylalaninemia

Classic tetrahydrobiopterin (BH(4)) deficiencies are characterized by hyperphenylalaninemia and deficiency of monoamine neurotransmitters. In this article, we report two patients with progressive psychomotor retardation, dystonia, severe dopamine and serotonin deficiencies (low levels of 5-hydroxyindoleacetic and homovanillic acids), and abnormal pterin pattern (high levels of biopterin and dihydrobiopterin) in cerebrospinal fluid. Furthermore, they presented with normal urinary pterins and without hyperphenylalaninemia. Investigation of skin fibroblasts revealed inactive sepiapterin reductase (SR), the enzyme catalyzing the final two-step reaction in the biosynthesis of BH(4). Mutations in the SPR gene were detected in both patients and their family members. One patient was homozygous for a TC-->CT dinucleotide exchange, predicting a truncated SR (Q119X). The other patient was a compound heterozygote for a genomic 5-bp deletion (1397-1401delAGAAC) resulting in abolished SPR-gene expression and an A-->G transition leading to an R150G amino acid substitution and to inactive SR as confirmed by recombinant expression. The absence of hyperphenylalaninemia and the presence of normal urinary pterin metabolites and of normal SR-like activity in red blood cells may be explained by alternative pathways for the final two-step reaction of BH(4) biosynthesis in peripheral and neuronal tissues. We propose that, for the biosynthesis of BH(4) in peripheral tissues, SR activity may be substituted by aldose reductase (AR), carbonyl reductase (CR), and dihydrofolate reductase, whereas, in the brain, only AR and CR are fully present. Thus, autosomal recessive SR deficiency leads to BH(4) and to neurotransmitter deficiencies without hyperphenylalaninemia and may not be detected by neonatal screening for phenylketonuria.     

ColorHOR--novel graphical algorithm for fast scan of alpha satellite higher-order repeats and HOR annotation for GenBank sequence of human genome

MOTIVATION: GenBank data are at present lacking alpha satellite higher-order repeat (HOR) annotation. Furthermore, exact HOR consensus lengths have not been reported so far. Given the fast growth of sequence databases in the centromeric region, it is of increasing interest to have efficient tools for computational identification and analysis of HORs from known sequences. RESULTS: We develop a graphical user interface method, ColorHOR, for fast computational identification of HORs in a given genomic sequence, without requiring a priori information on the composition of the genomic sequence. ColorHOR is based on an extension of the key-string algorithm and provides a color representation of the order and orientation of HORs. For the key string, we use a robust 6 bp string from a consensus alpha satellite and its representative nature is tested. ColorHOR algorithm provides a direct visual identification of HORs (direct and/or reverse complement). In more detail, we first illustrate the ColorHOR results for human chromosome 1. Using ColorHOR we determine for the first time the HOR annotation of the GenBank sequence of the whole human genome. In addition to some HORs, corresponding to those determined previously biochemically, we find new HORs in chromosomes 4, 8, 9, 10, 11 and 19. For the first time, we determine exact consensus lengths of HORs in 10 chromosomes. We propose that the HOR assignment obtained by using ColorHOR be included into the GenBank database.