Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Analysis of recognition of transfer-messenger RNA by the ribosomal decoding center

Bacterial ribosomes stalled on defective mRNAs are rescued by tmRNA that functions as both tRNA and mRNA. The first ribosomal elongation cycle on tmRNA where tmRNA functions as tRNA is highly unusual: occupation of the ribosomal A site by tmRNA occurs without codon:anticodon pairing. Our analysis shows that in this case the role of a codon:anticodon duplex should be accomplished by a single unpaired triplet. In order that tmRNA could participate in the ribosomal elongation cycle, a triplet preceding the mRNA portion of tmRNA (the -1triplet) should be in the A-form and this form should be recognized by the ribosomal decoding center. A rule is derived that determines what triplets cannot be used as the -1triplet. The rule was tested with the -1triplets of all known 414 tmRNA species. All 23 observed -1triplets follow the formulated rule. The rule is also supported by the available data on base substitutions within the -1triplet.     

Erythrobacter vulgaris sp. nov., a novel organism isolated from the marine invertebrates

Four yellow-pigmented, gram-negative, chemoorganotrophic aerobic bacteria were isolated from starfish Stellaster equestris (strains 022-2-10T, 022-2-9, and 022-2-12) and soft coral (unidentified species) (strain 022-4-7) collected in the South China Sea. 16S rRNA gene sequence-based analyses of the new organisms revealed that Erythrobacter spp. were the closest relatives and shared the highest similarity of 98.7% to E. citreus, 98.5% to E. flavus, 97.9% to E. litoralis and 97.6% to E. longus. The novel organisms were tolerant to 3-6% NaCl, grew between 10 degrees C and 40 degrees C, and were not able to degrade gelatin, casein, and agar, while degraded Tween 80. Two strains (022-2-9 and 022-2-12) could weakly degrade starch. All strains produced a large pool of carotenoids and did not have Bacteriochlorophyll a. Phosphatidylethanolamine (30-36%), phosphatidylglycerol (39-46%), and phosphatidylcholine (21-27%) were the predominant phospholipids. Sphingoglycolipid was not detected. The major fatty acids were 16:0 (6-11%), 16:1omega7 (12-15%), and 18:1omega7 (46-49%). The two-hydroxy fatty acids, 13:0-2OH, 14:0-2OH, 15:0-2OH, 16:0-2OH were also present. The G + C content of the DNAs ranged from 61 to 62 mol%. The level of DNA similarity among four strains was conspecific and ranged from 94% to 98%. Even though new strains and other species of the genus had rather high level of 16S rRNA gene sequence similarities, DNA-DNA hybridization experiments showed only 33-39% of binding with the DNA of the type strains. On the basis of these results and the significant differences demonstrated in the phenotypic and chemotaxonomic characteristics, it is suggested that the new organisms be classified as a novel species; the name Erythrobacter vulgaris sp. nov. is proposed. The type strain is 022-2-10T (= KMM 3465T = CIP 107841T).     

Substances isolated from Mandragora species

The present state of knowledge in the chemistry of mandragora plant is reviewed. Isolations and identifications of the compounds were done from all parts of this plant. Up-to-date more than 80 substances were identified in different species of the genus Mandragora.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.     

19F NMR measurements of NO production in hypertensive ISIAH and OXYS rats

Recently we demonstrated the principal possibility of application of 19F NMR spin-trapping technique for in vivo *NO detection [Free Radic. Biol. Med. 36 (2004) 248]. In the present study, we employed this method to elucidate the significance of *NO availability in animal models of hypertension. In vivo *NO-induced conversion of the hydroxylamine of the fluorinated nitronyl nitroxide (HNN) to the hydroxylamine of the iminonitroxide (HIN) in hypertensive ISIAH and OXYS rat strains and normotensive Wistar rat strain was measured. Significantly lower HIN/HNN ratios were measured in the blood of the hypertensive rats. The NMR data were found to positively correlate with the levels of nitrite/nitrate evaluated by Griess method and negatively correlate with the blood pressure. In comparison with other traditionally used methods 19F NMR spectroscopy allows in vivo evaluation of *NO production and provides the basis for in vivo *NO imaging.     

Molecular Characteristics of OmpF-Like Porins from Pathogenic <Emphasis Type="Italic">Yersinia</Emphasis>

Nonspecific pore-forming proteins (porins) are the major proteins of the outer membrane of Gram-negative bacteria responsible for diffusion of low-molecular-weight compounds. Nucleotide sequences of the OmpF-like porins from the pathogenic bacteria Yersinia pseudotuberculosis (YPS) and Yersinia enterocolitica (YE) were cloned and determined. Values of molecular weights (MW) and isoelectric points (IEP) calculated for these proteins (for OmpF-YPS: MW 37.7 kD, IEP 4.45; for OmpF-YE: MW 39.5 kD, IEP 4.34) are in good agreement with experimental data. The OmpF-like Yersinia porins are highly homologous to each other (83-92%) and also to the OmpF protein from Serratia marcescens (70%); the homology to the OmpF porin from E. coli is significantly lower (52-58%). Multiple alignment of the amino acid sequences of mature OmpF proteins provided the distribution of conservative amino acid residues typical for porins. Moreover, the OmpF-like porins from Yersinia are characterized by the presence of extended regions with high and low homologies, which coincide with the transmembrane domains and "external" loops, respectively, of the topological model of the OmpF porin from E. coli. By predictive methods, the secondary structure of the OmpF-like porins from Yersinia was obtained. This structure is represented by 16 beta-strands connected by short "periplasmic" and longer "external" loops with unordered structure.     

Glass-forming property of the system diethyl sulphoxide/water and its cryoprotective action on Escherichia coli survival

In this work the thermal properties of diethyl sulphoxide (Et2SO), as well as its cryoprotective ability are studied and related to other well-known cryoprotectant substances, like dimethyl sulphoxide (Me2SO). We have investigated the thermal properties of Et2SO/water systems using Differential Scanning Calorimetry at a very low heating/cooling rate (2 degrees C/min). Liquid/solid or glassy/crystalline transitions have been observed only for the solutions with content of Et2SO ranging from 5 up to 40% w/w and/or greater than 85%. In the 45-75% w/w Et2SO range we have found a noticeable glass-forming tendency and a great stability of the amorphous state to the reheating. In samples with Et2SO content ranging from 80 to 85%, we observed a great stability of the glass forming by cooling, but a lesser stability to the subsequent reheating. The glass-forming tendency of these solutions is discussed in terms of existing competitive interactions between molecules of Et2SO, on the one hand, and Et2SO and water molecules, on the other hand. The results are well explainable on the basis of the model structure of water/Et2SO solutions, deduced by Raman and infrared studies [J. Mol. Struct. 665 (2003) 285-292]. The cryoprotective ability of Et2SO on Escherichia coli survival has been also investigated, and a comparison among Et2SO and other widely used cryoprotectants, like Me2SO and glycerol has been done. Survival of E. coli, determined after freezing-thawing process, was maximal at 45% w/w Et2SO (more than 85% viability). It should be noted that at the same concentration the survival is only about 35% in the presence of Me2SO and not more than 15% in the presence of glycerol. These features are well consisted with the glass-forming properties of Et2SO.     

Identification and expression of two novel cytochrome P450 genes, belonging to CYP4 and a new CYP331 family, in the polychaete Capitella capitata sp.I

The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily.     

Overlapping Messages and Survivability

The phenomenon of overlapping of various sequence messages in genomes is a puzzle for evolutionary theoreticians, geneticists, and sequence researchers. The overlapping is possible due to degeneracy of the messages, in particular, degeneracy of codons. It is often observed in organisms with a limited size of genome, possessing polymerases of low fidelity. The most accepted view considers the overlapping as a mechanism to increase the amount of information per unit length. Here we present a model that suggests direct evolutionary advantage of the message overlapping. Two opposing drives are considered: (a) reduction in the amount of vulnerable points when the overlapping of two messages involves common critical points and (b) cumulative compromising cost of coexistence of messages at the same site. Over a broad range of conditions the reduction of the target size prevails, thus making the overlapping of messages advantageous.[Reviewing Editor: Dr. Martin Kreitman]