Guanidination of Soluble Lysine-Rich Cyanophycin Yields a Homoarginine-Containing Polyamide

Soluble cyanobacterial granule polypeptide (CGP), especially that isolated from recombinant Escherichia coli strains, consists of aspartic acid, arginine, and a greater amount of lysine than that in insoluble CGP isolated from cyanobacteria or various other recombinant bacteria. In vitro guanidination of lysine side chains of soluble CGP with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. The modified soluble CGP consisted of 51 mol% aspartate, 14 mol% arginine, and 35 mol% homoarginine. The complete conversion of lysine residues to homoarginine was confirmed by (i) nuclear magnetic resonance spectrometry, (ii) coupled liquid chromatography-mass spectrometry, and (iii) high-performance liquid chromatography. Unlike soluble CGP, this new homoarginine-containing polyamide was soluble only under acidic or alkaline conditions and was insoluble in water or at a neutral pH. Thus, it showed solubility behavior similar to that of the natural insoluble polymer isolated from cyanobacteria, consisting of aspartic acid and arginine only. Polyacrylamide gel electrophoresis revealed similar degrees of polymerization of the native (12- to 40-kDa) and modified (10- to 35-kDa) polymers. This study showed that the chemical structure and properties of a biopolymer could be changed by in vitro introduction of a new functional group after biosynthesis of the native polymer. In addition, the modified CGP could be digested in vitro using the cyanophycinase from Pseudomonas alcaligenes strain DIP1, yielding a new dipeptide consisting of aspartate and homoarginine.     

Mutations in the N-terminal actin-binding domain of filamin C cause a distal myopathy

Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations.     

The cross-road between the mechanisms of protein folding and aggregation; study of human stefin B and its H75W mutant

The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the “molten globule” type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.     

Ca(2+) sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca(2+) concentration is necessary and sufficient for this process. The predominant source of Ca(2+) for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca(2+) to the cytosol. The ER store is (re)filled by the store-specific Ca(2+)-ATPase. Ultimately, the depleted ER is replenished by Ca(2+) which enters from the extracellular space to the cytosol via store-operated Ca(2+) entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca(2+) channels and plasma membrane Na(+)/Ca(2+) exchangers are additional means for cytosolic Ca(2+) entry. Cytosolic Ca(2+) levels can be modulated by mitochondria, which can take up cytosolic Ca(2+) via the Ca(2+) uniporter and release Ca(2+) into cytosol via the mitochondrial Na(+)/Ca(2+) exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca(2+) sources generates cytosolic Ca(2+) dynamics that can drive Ca(2+)-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Morphometric and molecular (RAPD) analysis of six <Emphasis Type="Italic">Serapias</Emphasis> taxa from Croatia

The morphometric analyses and genetic variability assessed by RAPD markers have been used to analyse relations among six Serapias taxa from Croatia (S. istriaca, S. pulae originally described as hybrid, S. ionica, S. vomeracea, S. lingua and S. cordigera). S. istriaca distributed in southern Istria and the island of Lošinj and S. pulae stenoendemic taxon distributed only in southern Istria S. ionica is endemic to the Ionian and Dalmatian islands, while the remaining taxa are more widely distributed. The obtained results shows that the endemic S. istriaca is a well characterised taxon, that S. pulae is a hybrid between S. istriaca and S. lingua and that the hybrid is morphologically and genetically more similar to S. lingua than the second parental species S. istriaca. The division into the subsections Steno-, Medio- and Platypetalae is founded based on the floral morphology while the division into the sections Serapias and Bilamellaria is not evident in the quantitative morphological and genetic analyses. Furthermore, considerable genetic resemblance between S. vomeracea and S. ionica was established.     

The flight distances of floodwater mosquitoes (<Emphasis Type="Italic">Aedes vexans,Ochlerotatus sticticus</Emphasis> and <Emphasis Type="Italic">Ochlerotatus caspius</Emphasis>) in Osijek,Eastern Croatia

In spring 2004, the mark-release-recapture study was conducted in the Osijek area, covering the total of 171 km², to describe dispersal pattern of three floodwater mosquito species (Aedes vexans, Ochlerotatus sticticus and Oc. caspius). Forty CO₂ baited Center for Disease Control and Prevention (CDC) traps were set at distances varying from 0.95 to 16.7 km from release site. Fifty thousand mosquitoes were released, and a total of 22 marked mosquitoes were recaptured in 12 traps, 82% of which were collected in the first six days after release. The maximum distance of recaptured mosquito (Oc. sticticus) was found at 11.68 km from the release site. During the study, the average dispersal rate per day for mosquitoes ranged from 0.96 km to 3.6 km in regard to different species.     

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k_off, compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.     

Eimeria papillata: upregulation of specific miRNA-species in the mouse jejunum

Increasing evidence indicates miRNAs as critical regulators of gene expression, but little information is available for miRNAs in intestinal diseases. Here, we investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, mice were shedding 3150 ± 430 oocysts per gram feces. This was associated with a low inflammatory response of the jejunum of mice evidenced by histology, non-response of IL-1β mRNA, even slight downregulation of IL-6 mRNA, only slight increases in iNOS mRNA, nitrate/nitrate, malondialdehyde, and a small decrease in glutathione, respectively. Only IFNγ mRNA was strongly induced. Using miRNA microarray technology, there were significantly upregulated the four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. Our data provide evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ.     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.